Purinergic Stimulation Research Articles

Protective Role of Cardiac CFTR Activation Upon Early Reperfusion Against Myocardial Infarction

Background: The cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR) was shown to be activated by β-adrenergic or purinergic stimulation and involved in cell volume regulation after osmotic swelling. Also, cardiac CFTR was reported to be essential in the mechanism by which ischemic preconditioning protects against ischemia/reperfusion(I/ R)-induced injury of the heart. Here, we explored the possibility that activation of cardiac CFTR can provide protection against I/R-induced myocardial infarction, even after ischemic attack. Methods: The hearts of wild-type mice were subjected to 30- or 40-min left coronary artery occlusion followed by 2-h or 2-day reperfusion in vivo, and myocardial infarction was examined under a variety of conditions. Neonetal rat ventricular myocytes in primary culture were subjected to hypoxia/reoxygenation in vitro, and necrotic cell death was examined. Results: The infarct size was much greater in CFTR knockout mice than in wild-type mice. Intravenous infusion of a number of putative CFTR activators upon reperfusion prominently reduced the size of myocardial infarction in wild-type but not CFTR-deficient mice. This protective effect was abolished by co-administration of a CFTR inhibitor. CFTR activators ameliorated, in a manner sensitive to a CFTR inhibitor, release of myocardial-specific creatine kinase isoenzyme to the serum in mice subjected to I/R in vivo. Necrotic death of cultured neonatal rat ventricular myocytes subjected to hypoxia/reoxygenation in vitro was ameliorated by CFTR activators or CFTR gene overexpression but aggravated by a CFTR inhibitor or CFTR gene silencing. Conclusion: It is concluded that activation of endogenous myocardial CFTR upon early reperfusion is involved in protection against necrotic myocardial injury induced by I/R in vivo and in vitro. Cardiac CFTR may serve as a target accessible even after ischemic attack for pharmacotherapeutic intervention in I/R-induced myocardial infarction.

Adenosine triphosphate (ATP) inhibits IP3-evoked Ca2+ release in smooth muscle via P2Y1 receptors

SummaryAdenosine 5′-triphosphate (ATP) mediates a variety of biological functions following nerve-evoked release, via activation of either G-protein-coupled P2Y- or ligand-gated P2X receptors. In smooth muscle, ATP, acting via P2Y receptors (P2YR), may act as an inhibitory neurotransmitter. The underlying mechanism(s) remain unclear, but have been proposed to involve the production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] by phospholipase C (PLC), to evoke Ca2+ release from the internal store and stimulation of Ca2+-activated potassium (KCa) channels to cause membrane hyperpolarization. This mechanism requires Ca2+ release from the store. However, in the present study, ATP evoked transient Ca2+ increases in only ~10% of voltage-clamped single smooth muscle cells. These results do not support activation of KCa as the major mechanism underlying inhibition of smooth muscle activity. Interestingly, ATP inhibited Ins(1,4,5)P3-evoked Ca2+ release in cells that did not show a Ca2+ rise in response to purinergic activation. The reduction in Ins(1,4,5)P3-evoked Ca2+ release was not mimicked by adenosine and therefore, cannot be explained by hydrolysis of ATP to adenosine. The reduction in Ins(1,4,5)P3-evoked Ca2+ release was, however, also observed with its primary metabolite, ADP, and blocked by the P2Y1R antagonist, MRS2179, and the G protein inhibitor, GDPβS, but not by PLC inhibition. The present study demonstrates a novel inhibitory effect of P2Y1R activation on Ins(1,4,5)P3-evoked Ca2+ release, such that purinergic stimulation acts to prevent Ins(1,4,5)P3-mediated increases in excitability in smooth muscle and promote relaxation.

Inhibition of the Ca2+-Dependent K+Channel,KCNN4/KCa3.1, Improves Tissue Protection and Locomotor Recovery after Spinal Cord Injury

Spinal cord injury (SCI) triggers inflammatory responses that involve neutrophils, macrophages/microglia and astrocytes and molecules that potentially cause secondary tissue damage and functional impairment. Here, we assessed the contribution of the calcium-dependent K⁺ channel KCNN4 (KCa3.1, IK1, SK4) to secondary damage after moderate contusion lesions in the lower thoracic spinal cord of adult mice. Changes in KCNN4 mRNA levels (RT-PCR), KCa3.1 protein expression (Western blots), and cellular expression (immunofluorescence) in the mouse spinal cord were monitored between 1 and 28 d after SCI. KCNN4 mRNA and KCa3.1 protein rapidly increased after SCI; double labeling identified astrocytes as the main cellular source accounting for this upregulation. Locomotor function after SCI, evaluated for 28 d in an open-field test using the Basso Mouse Scale, was improved in a dose-dependent manner by treating mice with a selective inhibitor of KCa3.1 channels, TRAM-34 (triarylmethane-34). Improved locomotor function was accompanied by reduced tissue loss at 28 d and increased neuron and axon sparing. The rescue of tissue by TRAM-34 treatment was preceded by reduced expression of the proinflammatory mediators, tumor necrosis factor-α and interleukin-1β in spinal cord tissue at 12 h after injury, and reduced expression of inducible nitric oxide synthase at 7 d after SCI. In astrocytes in vitro, TRAM-34 inhibited Ca²⁺ signaling in response to metabotropic purinergic receptor stimulation. These results suggest that blocking the KCa3.1 channel could be a potential therapeutic approach for treating secondary damage after spinal cord injury.

Purinergic activation of rat skeletal muscle membranes increases V max and Na+ affinity of the Na,K-ATPase and phosphorylates phospholemman and α1 subunits

Muscle activity is associated with an increase in extracellular purines (ATP, ADP), which are involved in signalling mechanisms. The present study investigates the effect of purines on the function of Na,K-ATPase (Na,K-pump) in rat skeletal muscle. Na,K-ATPase activity was quantified by measuring the release of inorganic phosphate in the presence of ATP and variable Na(+) concentrations. In membranes purified from glycolytic muscle fibres, purinergic stimulation increases V (max) and decreases the K (m) (higher Na(+) affinity) of the Na,K-ATPase. Stimulatory effects were obtained using ATP, ADP, 2-methylthio-ADP and UPT, but not UDP and adenosine. The effect of ADP on V (max) can be inhibited by the non-specific P2Y receptor antagonists, suramin and PPADS. Moreover, the P2Y(13) receptor antagonist MRS 2211 strongly inhibited the response to ADP, whereas the specific P2Y(1) receptor antagonist MRS 2500 had less effect. Based on results from these agonists and antagonists, we conclude that P2Y(13) receptors mediate the main effects observed, that P2Y1 receptors are also involved and that some P2Y(2)/P2Y(4) receptors also appear to be involved. Receptor antagonists had no effect on ADP-induced subunit (phospholemman and α1) phosphorylation and changes in K (m) (Na(+) affinity). Thus, the stimulatory effects of purines are mediated by two independent mechanisms: P2Y receptor-mediated increase in Na,K-ATPase capacity (increased V (max)) and P2Y receptor-independent phosphorylation of Na,K-ATPase phospholemman and α1 subunits, which induce changes in ion affinity. These mechanisms may contribute to up-regulation of Na,K-ATPase during muscle activity.

Reply from Scott M. O’Grady

The study by Palmer et al. (2011) is the third paper published from our laboratory on the characterization of a human mammary epithelial (HME) cell line that we immortalized by expression of the catalytic subunit of the human telomerase gene (Kim et al. 2002). In our previous study addressing the ion transport properties of these cells (Lee et al. 2007) we showed that the Na+ transport phenotype, P2Y receptor expression profile and lack of anion secretion observed in primary cells matched the results obtained from parallel studies conducted with immortalized HME cells. These findings stand in contrast to isolated mouse and bovine mammary epithelial cells that exhibit both ENaC-dependent Na+ transport and secretagogue-stimulated anion secretion (Blaug et al. 2001, 2003; Schmidt et al. 2001; Quesnell et al. 2007). In response to Dr Shennan's first comment on the origin of HME cells, as stated in our earlier study (Lee et al. 2007), we do not have conclusive proof that the cells originate from the duct. However, HME cells were obtained from a 51-year-old woman, an age where the process of lobular involution significantly reduces the number of secretory alveoli associated with the terminal portions of the ducts (Radisky & Hartmann, 2009). Moreover, lobular alveoli are thought to be the anatomic structures that give rise to breast cancer and in human mammary epithelial cancer cells (MCF-7 cells), purinergic stimulation increases CFTR-dependent anion transport (Flezar & Heisler, 1993). In contrast, HME cells do not exhibit anion secretion in response to purinergic receptor stimulation or to increases in intracellular Ca2+ or cAMP, and CFTR expression is extremely low; thus HME cells do not possess transport characteristics that are typically associated with fluid-secreting epithelia. However, ENaC-dependent Na+ absorption is characteristic of duct epithelial cells found in other exocrine glands such as salivary glands and sweat ducts (Cook et al. 2002; Reddy & Quinton, 2005). With respect to the second comment raised in Dr Shennan's letter, we would like to point out that the experiments described in Palmer et al. (2011) and Lee et al. (2007) were conducted with HME cells grown in media containing hydrocortisone, insulin, epidermal growth factor and bovine pituitary extract as a source for lactogenic hormones. Information about the composition of mammary epithelial growth media (MEGM) is explicitly stated in the Materials section of the manuscript. Our results showing that stimulation with ATP or UTP produces mobilization of intracellular Ca2+ is entirely consistent with what is known about P2Y receptor signalling pathways and is similar to the results obtained with ATP stimulation of MCF-7 cells (Flezar & Heisler, 1993). We suggest that the lack of Ca2+ mobilization following ATP stimulation in lactating mouse acini and bovine mammary cells referenced by Dr Shennan may reflect species differences that could be related to distinct P2Y receptor expression patterns and/or second messenger coupling. As for Dr Shenann's third comment about the previous studies by Linzell and Peaker (Linzell & Peaker, 1971a,b;), it is certainly possible that the transport characteristics of HME cells may not reflect the in vivo ion permeability properties of the duct epithelium. However, it is equally possible that the results obtained by Linzell and Peaker in goats do not necessarily represent the permeability characteristics of human duct epithelium. A possible application of the results obtained from our studies that might provide added insight about the permeability properties of human duct epithelium would be to use ENaC and KCa3.1 channel expression as possible biomarkers for duct epithelial cells. Future experiments could then employ immunohistochemical techniques using sections of human breast tissue to investigate the cellular distribution of these channels, which could be helpful in clarifying the role of the duct in determining the ionic composition of milk. The fourth comment in Dr Shennan's letter suggests an interesting experiment that we have not performed. It would not be surprising that lowering the ionic strength of the apical solution would produce an increase in transepithelial resistance since transcellular ion movements would be reduced under these conditions. However, there may also be effects on expression or activity of membrane transporters, or possibly the paracellular pathway, that could be important in determining the overall permeability of the duct epithelium. Therefore, we would agree that future experiments examining the effects of milk constituents such as lactose on transport function would be worthwhile and perhaps expand the utility of cultured cell models in understanding the regulation of epithelial transport mechanisms that contribute to the ionic composition of milk.

Purinergic Activation of Ca2+-Permeable TRPV4 Channels Is Essential for Mechano-Sensitivity in the Aldosterone-Sensitive Distal Nephron

Mechanical forces are known to induce increases of [Ca2+]i in the aldosterone-sensitive distal nephron (ASDN) cells to regulate epithelial transport. At the same time, mechanical stress stimulates ATP release from ASDN cells. In this study, we combined ratiometric Fura-2 based monitoring of [Ca2+]i in freshly isolated split-opened ASDN with targeted deletion of P2Y2 and TRPV4 in mice to probe a role for purinergic signaling in mediating mechano-sensitive responses in ASDN cells. ATP application causes a reproducible transient Ca2+ peak followed by a sustained plateau. Individual cells of the cortical collecting duct (CCD) and the connecting tubule (CNT) respond to purinergic stimulation with comparative elevations of [Ca2+]i. Furthermore, ATP-induced Ca2+-responses are nearly identical in both principal (AQP2-positive) and intercalated (AQP2-negative) cells as was confirmed using immunohistochemistry in split-opened ASDN. UTP application produces elevations of [Ca2+]i similar to that observed with ATP suggesting a dominant role of P2Y2-like receptors in generation of [Ca2+]i response. Indeed, genetic deletion of P2Y2 receptors decreases the magnitude of ATP-induced and UTP-induced Ca2+ responses by more than 70% and 90%, respectively. Both intracellular and extracellular sources of Ca2+ appeared to contribute to the generation of ATP-induced Ca2+ response in ASDN cells. Importantly, flow- and hypotonic-induced Ca2+ elevations are markedly blunted in P2Y2 −/− mice. We further demonstrated that activation of mechano-sensitive TRPV4 channel plays a major role in the sustained [Ca2+]i elevation during purinergic stimulation. Consistent with this, ATP-induced Ca2+ plateau are dramatically attenuated in TRV4 −/− mice. Inhibition of TRPC channels with 10 µM BTP2 also decreased ATP-induced Ca2+ plateau whilst to a lower degree than that observed with TRPV4 inhibition/genetic deletion. We conclude that stimulation of purinergic signaling by mechanical stimuli leads to activation of TRPV4 and, to a lesser extent, TRPCs channels, and this is an important component of mechano-sensitive response of the ASDN.

Adenosine activation of A2Breceptor(s) is essential for stimulated epithelial ciliary motility and clearance

Mucociliary clearance, vital to lung clearance, is dependent on cilia beat frequency (CBF), coordination of cilia, and the maintenance of periciliary fluid. Adenosine, the metabolic breakdown product of ATP, is an important modulator of ciliary motility. However, the contributions of specific adenosine receptors to key airway ciliary motility processes are unclear. We hypothesized that adenosine modulates ciliary motility via activation of its cell surface receptors (A(1), A(2A), A(2B), or A(3)). To test this hypothesis, mouse tracheal rings (MTRs) excised from wild-type and adenosine receptor knockout mice (A(1), A(2A), A(2B), or A(3), respectively), and bovine ciliated bronchial epithelial cells (BBECs) were stimulated with known cilia activators, isoproterenol (ISO; 10 μM) and/or procaterol (10 μM), in the presence or absence of 5'-(N-ethylcarboxamido) adenosine (NECA), a nonselective adenosine receptor agonist [100 nM (A(1), A(2A), A(3)); 10 μM (A(2B))], and CBF was measured. Cells and MTRs were also stimulated with NECA (100 nM or 10 μM) in the presence and absence of adenosine deaminase inhibitor, erythro-9- (2-hydroxy-3-nonyl) adenine hydrochloride (10 μM). Both ISO and procaterol stimulated CBF in untreated cells and/or MTRs from both wild-type and adenosine knockout mice by ~3 Hz. Likewise, CBF significantly increased ~2-3 Hz in BBECs and wild-type MTRs stimulated with NECA. MTRs from A(1), A(2A), and A(3) knockout mice stimulated with NECA also demonstrated an increase in CBF. However, NECA failed to stimulate CBF in MTRs from A(2B) knockout mice. To confirm the mechanism by which adenosine modulates CBF, protein kinase activity assays were conducted. The data revealed that NECA-stimulated CBF is mediated by the activation of cAMP-dependent PKA. Collectively, these data indicate that purinergic stimulation of CBF requires A(2B) adenosine receptor activation, likely via a PKA-dependent pathway.

CD36 Protein Is Involved in Store-operated Calcium Flux, Phospholipase A2 Activation, and Production of Prostaglandin E2

The scavenger receptor FAT/CD36 contributes to the inflammation associated with diabetes, atherosclerosis, thrombosis, and Alzheimer disease. Underlying mechanisms include CD36 promotion of oxidative stress and its signaling to stress kinases. Here we document an additional mechanism for the role of CD36 in inflammation. CD36 regulates membrane calcium influx in response to endoplasmic reticulum (ER) stress, release of arachidonic acid (AA) from cellular membranes by cytoplasmic phospholipase A(2)α (cPLA(2)α) and contributes to the generation of proinflammatory eicosanoids. CHO cells stably expressing human CD36 released severalfold more AA and prostaglandin E(2) (PGE(2)), a major product of AA metabolism by cyclooxygenases, in response to thapsigargin-induced ER stress as compared with control cells. Calcium influx after ER calcium release resulted in phosphorylation of cPLA(2) and its translocation to membranes in a CD36-dependent manner. Peritoneal macrophages from CD36(-/-) mice exhibited diminished calcium transients and reduced AA release after thapsigargin or UTP treatment with decreased ERK1/2 and cPLA(2) phosphorylation. However, PGE(2) production was unexpectedly enhanced in CD36(-/-) macrophages, which probably resulted from a large induction of cyclooxygenase 2 mRNA and protein. The data demonstrate participation of CD36 in membrane calcium influx in response to ER stress or purinergic receptor stimulation resulting in AA liberation for PGE(2) formation. Collectively, these results identify a mechanism contributing to the pleiotropic proinflammatory effects of CD36 and suggest that its targeted inhibition may reduce the acute inflammatory response.

Consequences of Epidermal Growth Factor Receptor (ErbB1) Loss for Vascular Smooth Muscle Cells From Mice With Targeted Deletion of ErbB1

Pathophysiological effects of the epidermal growth factor receptor (EGFR or ErbB1) include vascular remodeling. EGFR transactivation is proposed to contribute significantly to heterologous signaling and remodeling in vascular smooth muscle cells (VSMC). We investigated the importance of EGFR in primary VSMC from aorta of mice with targeted deletion of the EGFR (EGFR(Δ/Δ VSMC)→VSMC(EGFR-/-) and EGFR(Δ/+ VSMC)→VSMC(EGFR+/-)) and the respective littermate controls (EGFR(+/+ VSMC)→VSMC(EGFR+/+)) with respect to survival, pentose phosphate pathway activity, matrix homeostasis, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and Ca(2+) homeostasis. In VSMC(EGFR-/-), epidermal growth factor-induced signaling was abolished; VSMC(EGFR+/-) showed an intermediate phenotype. EGFR deletion enhanced spontaneous cell death, reduced pentose phosphate pathway activity, disturbed cellular matrix homeostasis (collagen III and fibronectin), and abolished epidermal growth factor sensitivity. In VSMC(EGFR-/-) endothelin-1- or α(1)-adrenoceptor-induced ERK1/2 phosphorylation and the fraction of Ca(2+) responders were significantly reduced, whereas responsive cells showed a significantly stronger Ca(2+) signal. Oxidative stress (H(2)O(2)) induced ERK1/2 activation in VSMC(EGFR+/+) and VSMC(EGFR+/-) but not in VSMC(EGFR-/-). The Ca(2+) signal was enhanced in VSMC(EGFR-/-), similar to purinergic stimulation by ATP. In conclusion, EGFR was found to be important for basal VSMC homeostasis and ERK1/2 activation by the tested G-protein-coupled receptors or radical stress. Ca(2+) signaling was modulated by EGFR differentially with respect to the fraction of responders and magnitude of the signal. Thus, EGFR seems to be Janus-faced for VSMC biology.

Influence of pyridylisatogen tosylate on contractions produced by ATP and by purinergic stimulation in the terminal ileum of the guinea-pig.

2-2'Pyridylisatogen tosylate (PIT), which antagonizes the inhibitory action of ATP in the taenia caeci, did not antagonize the excitatory effects of exogenous ATP and of purinergic stimulation of the terminal guinea-pig ileum. PIT (0-5--2-5 muM) potentiated the ATP-induced contractions and also the contractions produced by potassium chloride, though the potentiation could not be related to the dose in every experiment. The responses to noradrenaline, adrenaline and histamine were slightly inhibited. PIT also potentiated the contractions produced by electrical stimulation of intramural purinergic nerves when either an alternate or uniform stimulation pattern was used. The present results in which the preparation is contracted by ATP are opposite to those obtained with PIT on the taenia caeci, which is relaxed by ATP. This raises a question of duality or plurality of receptors for ATP.

Species Specificity of ADAM10 and ADAM17 Proteins in Interleukin-6 (IL-6) Trans-signaling and Novel Role of ADAM10 in Inducible IL-6 Receptor Shedding

Hypomorphic ADAM17(ex/ex) mice showed defects in mucosal regeneration due to inefficient enhanced GFR shedding. ADAM17 is the main sheddase of interleukin-6 receptor (IL-6R) to induce IL-6 trans-signaling. However, serum levels of soluble murine IL-6R were not reduced in ADAM17(ex/ex) mice, and murine ADAM17 was not the major sheddase of murine IL-6R. Shedding of murine IL-6R by murine ADAM17 was rescued in chimeric murine IL-6R proteins containing any extracellular domain but not the transmembrane and intracellular domain of human IL-6R. Apoptosis is a physiological stimulus of ADAM17-mediated shedding of human IL-6R. Even though apoptosis induced IL-6R shedding in mice, the responsible protease was identified as ADAM10. ADAM10 also was identified as protease responsible for ionomycin-induced shedding of murine and human IL-6R. However, in ADAM10-deficient murine embryonic fibroblasts, compensatory shedding of human IL-6R was mediated by ADAM17, but loss of ADAM10-mediated shedding of murine IL-6R was compensated by an as-yet-unidentified protease. Finally, we identified physiological purinergic P2X7 receptor stimulation as a novel inducer of murine and human IL-6R shedding solely mediated by ADAM10. In conclusion, we describe an unexpected species specificity of ADAM10 and ADAM17 and identified ADAM10 as novel inducible sheddase of IL-6R in mice and humans, which might have consequences for the interpretation of phenotypes from ADAM17- and ADAM10-deficient mice.

Proinflammatory treatment of astrocytes with lipopolysaccharide results in augmented Ca2+ signaling through increased expression of VIA phospholipase A2 (iPLA2)

Many Ca(2+)-regulated intracellular processes are involved in the development of neuroinflammation. However, the changes of Ca(2+) signaling in the brain under inflammatory conditions were hardly studied. ATP-induced Ca(2+) signaling is a central event of signal transmission in astrocytic networks. We investigated primary astrocytes after proinflammatory stimulation with lipopolysaccharide (LPS; 100 ng/ml) for 6-24 h. We reveal that Ca(2+) responses to purinergic ATP stimulation are significantly increased in amplitude and duration after stimulation with LPS. We detected that increased amplitudes of Ca(2+) responses to ATP in LPS-treated astrocytes can be explained by substantial increase of Ca(2+) load in stores in endoplasmic reticulum. The mechanism implies enhanced Ca(2+) store refilling due to the amplification of capacitative Ca(2+) entry. The reason for the increased duration of Ca(2+) responses in LPS-treated cells is also the amplified capacitative Ca(2+) entry. Next, we established that the molecular mechanism for the LPS-induced amplification of Ca(2+) responses in astrocytes is increased expression and activity of VIA phospholipase A(2) (VIA iPLA(2)). Indeed, both gene silencing with specific small interfering RNA and pharmacological inhibition of VIA iPLA(2) with S-bromoenol lactone reduced the load of the Ca(2+) stores and caused a decrease in the amplitudes of Ca(2+) responses in LPS-treated astrocytes to values, which were comparable with those in untreated cells. Our findings highlight a novel regulatory role of VIA iPLA(2) in development of inflammation in brain. We suggest that this enzyme might be a possible target for treatment of pathologies related to brain inflammation.

Purinergic Receptor Stimulation Reduces Cytotoxic Edema and Brain Infarcts in Mouse Induced by Photothrombosis by Energizing Glial Mitochondria

Treatments to improve the neurological outcome of edema and cerebral ischemic stroke are severely limited. Here, we present the first in vivo single cell images of cortical mouse astrocytes documenting the impact of single vessel photothrombosis on cytotoxic edema and cerebral infarcts. The volume of astrocytes expressing green fluorescent protein (GFP) increased by over 600% within 3 hours of ischemia. The subsequent growth of cerebral infarcts was easily followed as the loss of GFP fluorescence as astrocytes lysed. Cytotoxic edema and the magnitude of ischemic lesions were significantly reduced by treatment with the purinergic ligand 2-methylthioladenosine 5′ diphosphate (2-MeSADP), an agonist with high specificity for the purinergic receptor type 1 isoform (P2Y1R). At 24 hours, cytotoxic edema in astrocytes was still apparent at the penumbra and preceded the cell lysis that defined the infarct. Delayed 2MeSADP treatment, 24 hours after the initial thrombosis, also significantly reduced cytotoxic edema and the continued growth of the brain infarction. Pharmacological and genetic evidence are presented indicating that 2MeSADP protection is mediated by enhanced astrocyte mitochondrial metabolism via increased inositol trisphosphate (IP3)-dependent Ca2+ release. We suggest that mitochondria play a critical role in astrocyte energy metabolism in the penumbra of ischemic lesions, where low ATP levels are widely accepted to be responsible for cytotoxic edema. Enhancement of this energy source could have similar protective benefits for a wide range of brain injuries.

Purinergic stimulation of human mesenchymal stem cells potentiates their chemotactic response to CXCL12 and increases the homing capacity and production of proinflammatory cytokines

Extracellular adenosine triphosphate (ATP) is a well-recognized mediator of cell-to-cell communication. Here we show ATP effects on bone marrow (BM)-derived human mesenchymal stem cell (hMSCs) functions. ATP-induced modification of hMSCs gene expression profile was assessed by Affymetrix technology. Clonogenic and migration assays in vitro, as well as xenotransplant experiments in vivo, were performed to evaluate the effects of ATP on hMSCs proliferation and BM homing. Enzyme-linked immunosorbent assays were used to assess hMSCs cytokines production, whereas T-cell cultures demonstrated the immunoregulatory activity of ATP-treated hMSCs. hMSCs were resistant to the cytotoxic effects of ATP, as demonstrated by the lack of morphological and mitochondrial changes or release of intracellular markers of cell death. Gene expression profiling revealed that ATP-stimulated hMSCs underwent a downregulation of genes involved in cell proliferation, whereas those involved in cell migration were strongly upregulated. The inhibitory activity of ATP on hMSCs proliferation was confirmed by assessing clonogenic stromal progenitors. ATP potentiated the chemotactic response of hMSCs to the chemokine CXCL12, and increased their spontaneous migration. In vivo, the homing capacity of hMSCs to the BM of immunodeficient mice was significantly increased by pretreatment with ATP. Moreover, ATP increased the production of the proinflammatory cytokines interleukin-2, interferon-γ, and interleukin-12p70, while decreasing the anti-inflammatory cytokine interleukin-10, and this finding was associated with the reduced ability of MSCs to inhibit T-cell proliferation. Our data show that purinergic signaling modulates hMSCs functions and highlights a role for extracellular nucleotides in hMSCs biology.

Purinergic Stimulation of Human Bone Marrow-Derived Mesenchymal Stem Cells Modulate Their Function and Differentiation Potential.

AbstractAbstract 3848 Introduction:Human bone marrow derived Mesenchymal Stem Cells (hMSCs) are adult multipotent cells. hMSCs differentiate in vitro and in vivo into several tissue lineages originating from the three germinal layers making them attractive candidates for bioengineering and cellular therapy. Thus, it seems of great relevance to search putative messengers and signalling able to modulate their proliferation and differentiation. Nucleotides triphosphates are extracellular messengers binding to specific receptors (P2Rs) that modulate cell functions depending on the cell type. Controversial information is available on P2 expression and activity in hMSCs. Methods and Results:Here we found that hMSCs expressed several P2R subtypes. hMSCs were very resistant to the cytotoxic effects of high concentrations of ATP, as demonstrated by the lack of morphological and mitochondrial changes or release of intracellular markers of cell death. Gene expression profiling revealed that ATP treatment down-regulated cell proliferation and up-regulated cell migration genes in hMSCs. Functional studies confirmed the inhibitory activity of ATP on proliferation and clonogenic ability of hMSCs. Furthermore, ATP potentiated the chemotactic response of hMSCs to the chemokine CXCL12, and increased their spontaneous migration. In vivo, xenotransplant experiments showed that the homing capacity of hMSCs to murine bone marrow was increased by ATP pre-treatment. Moreover, ATP increased pro-inflammatory cytokines production (IL-2, IFN-g, IL-12p70), while decreased secretion of the anti-inflammatory cytokine IL-10. This finding was associated with the reduced ability of ATP-treated hMSC of inhibiting T-cell proliferation. Microarrays data suggested that several genes implicated in hMSC differentiation can be modulated by ATP treatment. To further investigate this issue, hMSCs cells were cultured under adipogenic or osteogenic conditions and were transiently exposed to ATP before starting differentiation or continuously exposed to ATP for the first 3 days of differentiation induction. We demonstrated that adipogenesis-related accumulation of lipids, analyzed by Oil red O staining, was more evident in ATP treated cultures. Furthermore, quantitative real time PCR (qRT-PCR) assay showed that mRNA expression of PPARg, a transcription factor early up-regulated during adipogenesis, was significantly increased in hMSCs differentiated cells treated with ATP. In osteogenic condition, analysis of mineralized area through Alizarin Red staining, indicated that ATP treatment enhanced the extent of mineralization compared to untreated control. The expression of RUNX2, a key transcription factor in osteogenesis, analyzed by qRT-PCR in differentiated cells confirmed data obtained in Alizarin-based assay. Conclusions:These data demonstrated that purinergic signalling modulates biological functions and differentiation potential of hMSCs. Disclosures:No relevant conflicts of interest to declare.

Mg2+ ions reduce microglial and THP-1 cell neurotoxicity by inhibiting Ca2+ entry through purinergic channels

Mg2+ is a known antagonist of some Ca2+ ion channels. It may therefore be able to counteract the toxic consequences of excessive Ca2+ entry into immune-type cells. Here we examined the effects of Mg2+ on inflammation induced by Ca2+ influx into microglia and THP-1 cells following activation of purinergic receptors. Using tissue culture, an inflammatory response was induced by treatment with either the P2X7 purinergic receptor agonist 2′,3′-[benzoyl-4-benzoyl]-ATP (BzATP) or the P2Y2,4 receptor agonist uridine 5′-triphosphate (UTP). Both microglia and THP-1 cells expressed the mRNAs for these receptors. Treatment produced a rapid rise in intracellular Ca2+ which was significantly reduced by Mg2+ or the calcium chelator BAPTA-AM. Purinergic receptor stimulation activated the intracellular inflammatory pathway P38 MAP kinase and NFκB. This caused release of TNFα, IL-6, nitrite ions and other materials that are neurotoxic to SH-SY5Y cells. These effects were all ameliorated by Mg2+. They were also partly ameliorated by the P2X7R antagonists, oxATP and KN-62, the P2YR antagonist MRS2179, and the store operated Ca2+ channel blocker, SK96365. These results indicate that elevated Mg2+ is a broad spectrum inhibitor of Ca2+ entry into microglia or THP-1 cells. Mg2+ administration may be a strategy for reducing the damaging consequences Ca2+ induced neuroinflammation in degenerative neurological disorders such as Alzheimer disease and Parkinson disease.

The platinum (II) complex [Pt(O,O′-acac)(γ-acac)(DMS)] alters the intracellular calcium homeostasis in MCF-7 breast cancer cells

It was previously demonstrated that [Pt(O,O′-acac)(γ-acac)(DMS)] exerted toxic effects at high doses, whilst sub-cytotoxic concentrations induced anoikis and decreased cell migration. Aim of this study was to investigate the hypothesis that [Pt(O,O′-acac)(γ-acac)(DMS)] alters the [Ca 2+] i and that this is linked to its ability to trigger rapid apoptosis in MCF-7 cells. Thus, cells were treated with [Pt(O,O′-acac)(γ-acac)(DMS)] and its effects on some of the systems regulating Ca 2+ homeostasis were studied, also in cells dealing with the complex changes occurring during the Ca 2+ signalling evoked by extracellular stimuli. [Pt(O,O′-acac)(γ-acac)(DMS)] caused the decrease of PMCA activity (but not SERCA or SPCA) and Ca 2+ membrane permeability. These two opposite effects on [Ca 2+] i resulted in its overall increase from 102 ± 12 nM to 250 ± 24 nM after 15 min incubation. The effects of [Pt(O,O′-acac)(γ-acac)(DMS)] were also evident when cells were stimulated with ATP: the changes in Ca 2+ levels caused by purinergic stimulation resulted altered due to decreased PMCA activity and to the closure of Ca 2+ channels opened by purinergic receptor. Conversely, [Pt(O,O′-acac)(γ-acac)(DMS)] did not affect the store-operated Ca 2+ channels opened by thapsigargin or by ATP. [Pt(O,O′-acac)(γ-acac)(DMS)] provoked the activation of PKC-α and the production of ROS that were responsible for the Ca 2+ permeability and PMCA activity decrease, respectively. The overall effect of [Pt(O,O′-acac)(γ-acac)(DMS)] is to increase the [Ca 2+] i, an effect that is likely to be linked to its ability to trigger rapid apoptosis in MCF-7 cells. These data reinforce the notion that [Pt(O,O′-acac)(γ-acac)(DMS)] would be a promising drug in cancer treatment.

Aging differentially modifies agonist-evoked mouse detrusor contraction and calcium signals

Although aging-induced changes in urinary bladder neurotransmission have been studied in some detail, information regarding alterations in detrusor muscle is scanty and addresses only partial aspects of the myogenic response of detrusor. Rodent bladder aging shows several features similar to those reported in humans. The aim of this study was to characterize in aged mouse the alterations of detrusor muscle contraction and the putative underlying changes in Ca(2+) signals. We studied in vitro the myogenic contraction induced by agonists in detrusor strips from adult (3months old) or aged (23-25months old) mice. In addition, we determined the agonist-induced [Ca(2+)](i) signals by epifluorescence microscopy in fura-2 loaded isolated detrusor cells. Aging impaired the contractile response of bladder strips to cholinergic stimulation with bethanechol and to chemical depolarization with KCl-containing solutions. On the contrary, the response to purinergic stimulation (ATP) was enhanced. Aging also diminished the transient Ca(2+) signal evoked by bethanechol and the Ca(2+) influx induced by KCl in bladder strips. Treatments aimed to release calcium from intracellular stores (caffeine and a low level of ionomycin in Ca(2+)-free medium) showed that aging reduces the size of agonist-releasable stores. Similar to contraction, the mobilization of Ca(2+) by ATP was increased in aged cells. Therefore, the differential effects of aging on detrusor contraction are associated to alterations of [Ca(2+)](i) signals: the cholinergic inhibition is due to inhibition of voltage-operated Ca(2+) influx and reduction of the size of intracellular Ca(2+) stores, while the age-induced ATP response is accompanied by an enhanced Ca(2+) mobilization.

Adenosine A2 Receptor Presence and Synergy with Cholinergic Stimulation in Rabbit Lacrimal Gland

Purpose: Secretion from the lacrimal gland is an important part of the well-being of the eye, and a central part in the search for treatment of dry eye syndrome. Adenosine has stimulatory effects on the lacrimal gland, and can potentiate the effect of the cholinergic agonist carbachol (Cch). The aim of the present study is to investigate the presence of the adenosine A2 receptor subtypes A2A and A2B in the rabbit lacrimal gland, and to characterize their role in regulated acinar cell secretion.Methods: Expression of the receptors was investigated using reverse transcriptase-PCR (RT-PCR) and immunofluorescence, and secretion effects were studied using a secretion assay in isolated lacrimal gland acinar cells.Results: Presence of both receptors was detected by RT-PCR and immunofluorescence. The secretion assay revealed a minor effect of stimulation of the A2 receptors, and a strong synergistic effect with the cholinergic agonist Cch. The synergistic effect was significantly reduced by the A2B antagonist PSB 1115, but not by the A2A antagonist SCH 58261, indicating that A2B is the receptor responsible for this potentiation.Conclusions: The study reveals the presence of the adenosine A2 receptor subtypes as well as a role for them in lacrimal gland secretion, and especially in the synergy with purinergic and cholinergic stimulation.

Intracellular Ca2+ responses and cell volume regulation upon cholinergic and purinergic stimulation in an immortalized salivary cell line

The water channel aquaporin 5 (AQP5) seems to play a key role in salivary fluid secretion and appears to be critical in the cell volume regulation of acinar cells. Recently, the cation channel transient potential vanilloid receptor 4 (TRPV4) was shown to be functionally connected to AQP5 and also to cell volume regulation in salivary glands. We used the Simian virus 40 (SV40) immortalized cell line SMG C10 from the rat submandibular salivary gland to investigate the effect of ATP and the neurotransmitter analogue carbachol on Ca(2+) signalling and cell volume regulation, as well as the involvement of TRPV4 in the responses. We used fura-2-AM imaging, cell volume measurements, and western blotting. Both carbachol and ATP increased the concentration of intracellular Ca(2+), but no volume changes could be measured. Inhibition of TRPV4 with ruthenium red impaired both ATP- and carbachol-stimulated Ca(2+) signals. Peak Ca(2+) signalling during hyposmotic exposure was significantly decreased following inhibition of TRPV4, while the cells' ability to volume regulate appeared to be unaffected. These results show that in the SMG C10 cells, simulation of nervous stimulation did not induce cell swelling, although the cells had intact volume regulatory mechanisms. Furthermore, even though Ca(2+) signals were not needed for this volume regulation, TRPV4 seems to play a role during ATP and carbachol stimulation.