Pannexin Channel Regulation of Cell Migration: Focus on Immune Cells.

The role of Pannexin (PANX) channels during collective and single cell migration is increasingly recognized. Amongst many functions that are relevant to cell migration, here we focus on the role of PANX-mediated adenine nucleotide release and associated autocrine and paracrine signaling. We also summarize the contribution of PANXs with the cytoskeleton, which is also key regulator of cell migration. PANXs, as mechanosensitive ATP releasing channels, provide a unique link between cell migration and purinergic communication. The functional association with several purinergic receptors, together with a plethora of signals that modulate their opening, allows PANX channels to integrate physical and chemical cues during inflammation. Ubiquitously expressed in almost all immune cells, PANX1 opening has been reported in different immunological contexts. Immune activation is the epitome coordination between cell communication and migration, as leukocytes (i.e., T cells, dendritic cells) exchange information while migrating towards the injury site. In the current review, we summarized the contribution of PANX channels during immune cell migration and recruitment; although we also compile the available evidence for non-immune cells (including fibroblasts, keratinocytes, astrocytes, and cancer cells). Finally, we discuss the current evidence of PANX1 and PANX3 channels as a both positive and/or negative regulator in different inflammatory conditions, proposing a general mechanism of these channels contribution during cell migration.

Alterations in the extracellular catabolism of nucleotides and platelet aggregation induced by high-fat diet in rats: effects of alpha-tocopherol

The aim of this study was to assess whether alfa-tocopherol administration prevented alterations in the ectonucleotidase activities and platelet aggregation induced by high-fat diet in rats. Thus, we examined four groups of male rats which received standard diet, high-fat diet (HFD), α-tocopherol (α-Toc), and high-fat diet plus α-tocopherol. HFD was administered ad libitum and α-Toc by gavage using a dose of 50 mg/kg. After 3 months of treatment, animals were submitted to euthanasia, and blood samples were collected for biochemical assays. Results demonstrate that NTPDase, ectonucleotide pyrophosphatase/phosphodiesterase, and 5'-nucleotidase activities were significantly decreased in platelets of HFD group, while that adenosine deaminase (ADA) activity was significantly increased in this group in comparison to the other groups (P < 0.05). When rats that received HFD were treated with α-Toc, the activities of these enzymes were similar to the control, but ADA activity was significantly increased in relation to the control and α-Toc group (P < 0.05). HFD group showed an increased in platelet aggregation in comparison to the other groups, and treatment with α-Toc significantly reduced platelet aggregation in this group. These findings demonstrated that HFD alters platelet aggregation and purinergic signaling in the platelets and that treatment with α-Toc was capable of modulating the adenine nucleotide hydrolysis in this experimental condition

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Historical perspective on the pathology of myocardial ischemia/reperfusion injury.

A selective history of the pathophysiological, structural, and metabolic changes found during an episode of severe myocardial ischemia in the canine heart is presented. The changes that cause ischemic injury to become irreversible are discussed in detail because these changes are the target of any successful therapy designed to prevent ischemic cell death. Of these, the disruption of the sarcolemma, an injury the development of which is accelerated in vivo by the contraction of viable tissue elsewhere in the heart traumatizing the ischemic area, plus the changes in high-energy phosphate and the total adenine nucleotide pool are considered to be the critical events leading to the development of irreversibility. The discovery of preconditioning with ischemia is discussed, together with a brief description of postconditioning. Finally, reperfusion injury is discussed in a summary fashion. The evidence for the fact that myocytes are salvaged by reperfusion is presented, as is the evidence that myocytes become unsalvageable by reperfusion as the duration of ischemia increases. The concept that some of the myocytes that die after successful reperfusion with arterial blood actually are killed by changes initiated by reperfusion, so-called lethal reperfusion injury, is attractive in that prevention of this change would lead to greater salvage; however, the prevalence of this phenomenon in clinical practice remains to be determined.

A new regulatory principle for in vivo biochemistry: pleiotropic low affinity regulation by the adenine nucleotides--illustrated for the glycolytic enzymes of Saccharomyces cerevisiae.

Enzymology tends to focus on highly specific effects of substrates, allosteric modifiers, and products occurring at low concentrations, because these are most informative about the enzyme's catalytic mechanism. We hypothesized that at relatively high in vivo concentrations, important molecular monitors of the state of living cells, such as ATP, affect multiple enzymes of the former and that these interactions have gone unnoticed in enzymology. We test this hypothesis in terms of the effect that ATP, ADP, and AMP might have on the major free-energy delivering pathway of the yeast Saccharomyces cerevisiae. Assaying cell-free extracts, we collected a comprehensive set of quantitative kinetic data concerning the enzymes of the glycolytic and the ethanol fermentation pathways. We determined systematically the extent to which the enzyme activities depend on the concentrations of the adenine nucleotides. We found that the effects of the adenine nucleotides on enzymes catalysing reactions in which they are not directly involved as substrate or product, are substantial. This includes effects on the Michaelis-Menten constants, adding new perspective on these, 100 years after their introduction.

AMP-activated protein kinase: nature's energy sensor.

Maintaining sufficient levels of ATP (the immediate source of cellular energy) is essential for the proper functioning of all living cells. As a consequence, cells require mechanisms to balance energy demand with supply. In eukaryotic cells the AMP-activated protein kinase (AMPK) cascade has an important role in this homeostasis. AMPK is activated by a fall in ATP (concomitant with a rise in ADP and AMP), which leads to the activation of catabolic pathways and the inhibition of anabolic pathways. Here we review the role of AMPK as an energy sensor and consider the recent finding that ADP, as well as AMP, causes activation of mammalian AMPK. We also review recent progress in structural studies on phosphorylated AMPK that provides a mechanism for the regulation of AMPK in which AMP and ADP protect it against dephosphorylation. Finally, we briefly survey some of the outstanding questions concerning the regulation of AMPK.

The relationship between oxygen and adenosine in astrocytic cultures.

Brain tissue oxygenation affects cerebral function and blood flow (CBF). Adenosine (Ado), a purine nucleoside, moderates neuronal activity, and arterial diameter. The cellular source of Ado in brain remains elusive; however, astrocytes are a logical site of production. Using astrocytic cultures, we tested the hypothesis that astrocytic derived Ado reflects cerebral oxygenation. We found that during alterations in pO(2), extracellular levels of Ado [Ado](e) changed rapidly. Graded reductions of oxygen tension revealed that[Ado](e) reached 10(-7) M to 10(-6) M with a pO(2) of 30-10mmHg, comparable with [Ado](e) and oxygen levels found in brain tissue during normoxemia. Higher O(2) levels were associated with a depression of [Ado](e). Under conditions of low pO(2) (pO(2)

[RNase L, a crucial mediator of innate immunity and other cell functions]. / La RNase L, un acteur essentiel de la réponse cellulaire antivirale.

The 2-5A/RNase L pathway is one of the first cellular defences against viruses. RNase L is an unusual endoribonuclease which activity is strictly regulated by its binding to a small oligonucleotide, 2-5A. 2-5A itself is very unusual, consisting of a series of 5'- triphosphorylated oligoadenylates with 2'-5' bonds. But RNase L activity is not limited to viral RNA cleavage. RNase L plays a central role in innate immunity, apoptosis, cell growth and differentiation by regulating cellular RNA stability and expression. Default in its activity leads to increased susceptibility to virus infections and to tumor development. RNase L gene has been identified as HPC1 (Hereditary Prostate Cancer 1) gene. Study of RNase L variant R462Q in etiology of prostate cancer has led to the identification of the novel human retrovirus closely related to xenotropic murine leukemia viruses (MuLVs) and named XMRV.

Inhibitory effects of adenine nucleotides and related substances on UDP-glucuronosyltransferase: structure-effect relationships and evidence for an allosteric mechanism.

The inhibitory effects of nucleotides and related substances on rat hepatic UDP-glucuronosyltransferase (UGT) were studied. ATP and NADP+ markedly reduced 4-methylumbelliferone (4-MU) UGT activity only when detergent-treated rat liver microsomes were used as the enzyme source. The IC50 values of adenine, ATP, NAD+ and NADP+ were estimated to be below 20 microM, whereas AMP had no inhibitory effect. From the kinetic behavior observed, these adenine-related compounds were assumed to inhibit UGT activity non-competitively without competing with either 4-MU or UDP-glucuronic acid. Among guanine, cytosine and their related nucleotides, only triphosphate nucleotides (CTP and GTP) exhibited potent UGT inhibition, although the effect of GTP was weak. Estradiol 3- and 17-glucuronidation were also inhibited by the inhibitors of 4-MU UGT. The only exception was that estradiol 17-glucuronidation activity was inhibited by AMP (IC50=31 microM). In addition, AMP antagonized the inhibitory effects of adenine, ATP, and NADP+ on 4-MU and estradiol 3- glucuronidation activities. These results suggest that (1) a number of cellular nucleotides present within the endoplasmic reticulum regulate UGT function; and (2) these substances bind to a common allosteric site on UGT to reduce catalytic function.

Rheostat control of gene expression by metabolites.

Organisms adapt to changes in environmental conditions by altering gene expression. Such homeostatic control is apparent in metabolism, where biosynthetic metabolites play a role in regulatory feedback loops. Increasing evidence shows that small-molecule metabolites also shape the structure of chromatin and directly regulate the transcription and translation processes. These endogenous metabolites bind specialized histones, are used as substrates by chromatin-modifying enzymes, regulate the activity of transcriptional corepressors, and even modulate the structure of RNA itself. In doing so, they act as dynamic rheostats that fine-tune the activity of hard-wired gene circuits. Metabolites emerge as key effectors in tweaking gene expression.

Adenylate-coupled ion movement. A mechanism for the control of nodule permeability to O2 diffusion.

In response to changes in phloem supply, adenylate demand, and oxygen status, legume nodules are known to exercise rapid (seconds to hours) physiological control over their permeability to oxygen diffusion. Diffusion models have attributed this permeability control to the reversible flow of water into or out of intercellular spaces. To test hypotheses on the mechanism of diffusion barrier control, nodulated soybean (Glycine max L. Merr.) plants were exposed to a range of treatments known to alter nodule O2 permeability (i.e. 10% O2, 30% O2, Ar:O2 exposure, and stem girdling) before the nodules were rapidly frozen, freeze dried, and dissected into cortex and central zone (CZ) fractions that were assayed for K, Mg, and Ca ion concentrations. Treatments known to decrease nodule permeability (30% O2, Ar:O2 exposure, and stem girdling) were consistently associated with an increase in the ratio of [K+] in cortex to [K+] in the CZ tissue, whereas the 10% O2 treatment, known to increase nodule permeability, was associated with a decrease in the [K+]cortex:[K+](CZ). When these findings were considered in the light of previous results, a proposed mechanism was developed for the adenylate-coupled movement of ions and water into and out of infected cells as a possible mechanism for diffusion barrier control in legume nodules.

Adenine nucleotides inhibit cytokine generation by human mast cells through a Gs-coupled receptor.

ATP and ADP activate functionally distinct G protein-coupled purinergic (P2Y) receptors. We determined the expression and function of adenine nucleotide-specific P2Y receptors on cord blood-derived human mast cells (hMCs). Human MCs expressed mRNA encoding the ADP-specific P2Y1, P2Y12, and P2Y13 receptors; the ATP/UTP-specific P2Y2 receptor; and the ATP-selective P2Y11 receptor. ADP (0.05-50 muM) induced calcium flux that was completely blocked by a P2Y1 receptor-selective antagonist and was not cross-desensitized by ATP. Low doses of ADP induced strong phosphorylation of ERK and p38 MAPKs; higher doses stimulated eicosanoid production and exocytosis. Although MAPK phosphorylation was blocked by a combination of P2Y1- and P2Y12-selective antagonists, neither interfered with secretion responses. Unexpectedly, both ADP and ATP inhibited the generation of TNF-alpha in response to the TLR2 ligand, peptidoglycan, and blocked the production of TNF-alpha, IL-8, and MIP-1beta in response to leukotriene D(4). These effects were mimicked by two ATP analogues, adenosine 5'-O-(3-thiotriphosphate) and 2',3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate (BzATP), but not by adenosine. ADP, ATP, adenosine 5'-O-(3-thiotriphosphate), and 2',3'-O-(4-benzoyl-benzoyl) adenosine 5'-triphosphate each induced cAMP accumulation, stimulated the phosphorylation of CREB, and up-regulated the expression of inducible cAMP early repressor, a CREB-dependent inhibitor of cytokine transcription. Human MCs thus express several ADP-selective P2Y receptors and at least one G(s)-coupled ADP/ATP receptor. Nucleotides could therefore contribute to MC-dependent microvascular leakage in atherosclerosis, tissue injury, and innate immunity while simultaneously limiting the extent of subsequent inflammation by attenuating the generation of inducible cytokines by MCs.

Platelet receptors for adenine nucleotides and thromboxane A2.

Adenosine diphosphate (ADP) and thromboxane A (2) (TXA (2)) are important physiological activators of platelets and exert their effects by acting on cell surface receptors. Platelet nucleotide receptors can be distinguished as three separate subtypes of the P2 receptor family. The P2X (1) receptor is a ligand-gated adenosine triphosphate (ATP) receptor that was originally mistaken for an ADP receptor. This calcium-influx-causing receptor mediates platelet shape change and plays an important role in thrombus formation in small arterioles. The P2Y (1) receptor, through activation of G (q) and phospholipase C, is required for ADP-induced platelet shape change, fibrinogen receptor activation, and TXA (2) generation. The G (i)-coupled P2Y (12) receptor plays an important role in platelet aggregation, potentiation of dense granule release, and TXA (2) generation. Both the P2Y receptors are crucial for in vivo thrombus formation. TXA (2) stimulates two subtypes of G protein-coupled TP receptor, TPalpha and TPbeta, but its effects in platelets are mediated predominantly through the alpha isoform. Although interference with the activation of G protein-coupled ADP or TP receptors results in increased bleeding times and protection from thromboembolism, TP receptor antagonists did not translate into effective antiplatelet drugs. Blockade of ADP receptor is a mode of newer classes of antithrombotic drugs in the coming era. This review focuses on the contribution of different nucleotide receptors and TP receptors to platelet function and their potential as antithrombotic agents.

Adenosine tetraphosphate, Ap4, a physiological regulator of intraocular pressure in normotensive rabbit eyes.

Adenosine 5' tetraphosphate, Ap(4), is a natural nucleotide present in many biological systems. This nucleotide has been found as a constituent of the nucleotide pool present in the aqueous humor of New Zealand rabbits. HPLC analysis confirmed its identity and calculated its concentration levels to be 197 +/- 21 nM. When applied topically to the rabbit eyes, this mononucleotide produced a reduction in the intraocular pressure, which was dose-dependent. The pD(2) value calculated from the dose-response curve was 7.28 +/- 0.47, which is equivalent to 52.48 nM. The time course of such intraocular pressure reduction presented a maximal decrease of IOP to 75.1 +/- 2.3% compared with the vehicle control value (100%), and the effect lasted for more than 2 h. Cross-desensitization studies demonstrated that Ap(4) effect was mediated via a P2X receptor in this system. P2 receptor antagonists suramin, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS), and reactive blue 2 (RB-2) showed that only the latter was able to revert the effect of Ap(4). Antagonists of adrenoceptors and cholinoceptors were able to partially reverse the effect of this nucleotide; this might indicate a connection with the neural mechanisms that control the intraocular pressure.

[Interferon-induced 2',5'-oligoadenylate system: key components and biological functions]. / Interferonindukovana systema 2',5'-olihoadenilatu: kliuchovi komponenty ta biolohichni funktsiï.

The current data about the 2',5'-oligoadenylate system is reviewed. Its role in interferon signaling and cell metabolism regulation is discussed. The interferon system is known to be characterized by a wide range of biological functions such as antiviral defense, control of cell growth and differentiation, oncogenic stability, apoptosis, immune activation, etc. The biological role of interferon that is the multifunctional cytokine is discussed more in detail. The structure of main components of interferon signal transduction cascade (2',5'-oligoadenylate, 2',5'-oligoadenylate-synthetase and ribonuclease L) is reviewed. The interferon-induced 2',5'-oligoadenylate system is considered as the component of common regulatory system coordinating cell metabolism.

Nucleotide-dependent triggering of RNA polymerase-DNA interactions by an AAA regulator of transcription.

Enhancer-dependent activator proteins, which act upon the bacterial RNA polymerase containing the sigma54 promoter specificity factor, belong to the AAA superfamily of ATPases. Activator-sigma54 contact is required for the sigma54-RNAP to isomerize and engage the DNA template for transcription. How ATP hydrolysis is used to trigger changes in sigma54-RNA polymerase and promoter DNA that lead to DNA opening is poorly understood. Here, band shift and footprinting assays were used to investigate the DNA binding activities of sigma54 and sigma54-RNA polymerase in the presence of the activator protein PspF bound to poorly hydrolysable analogues of ATP and the ATP hydrolysis transition-state analogue ADP.AlFx. Results show that different nucleotide-bound forms of PspF can change the interactions between sigma54, sigma54-RNA polymerase, and a DNA fork junction structure present within closed promoter complexes. This provides evidence that in the activation transduction pathway, several functional states of the activator, prior to ATP hydrolysis, can serve to alter the fork junction binding activity of sigma54 and sigma54-RNA polymerase that precede full DNA opening. A sequential set of nucleotide-dependent transitions in sigma54-RNA polymerase promoter complexes needed for productive open complex formation may therefore depend upon different nucleotide-bound forms of the activator.

Molecular basis for Kir6.2 channel inhibition by adenine nucleotides.

K(ATP) channels are comprised of a pore-forming protein, Kir6.x, and the sulfonylurea receptor, SURx. Interaction of adenine nucleotides with Kir6.2 positively charged amino acids such as K185 and R201 on the C-terminus causes channel closure. Substitution of these amino acids with other positively charged residues had small effects on inhibition by adenine nucleotide, while substitution with neutral or negative residues had major effects, suggesting electrostatic interactions between Kir6.2 positive charges and adenine nucleotide negative phosphate groups. Furthermore, R201 mutation decreased channel sensitivity to ATP, ADP, and AMP to a similar extent, but K185 mutation decreased primarily ATP and ADP sensitivity, leaving the AMP sensitivity relatively unaffected. Thus, channel inhibition by ATP may involve interaction of the alpha-phosphate with R201 and interaction of the beta-phosphate with K185. In addition, decreased open probability due to rundown or sulfonylureas caused an increase in ATP sensitivity in the K185 mutant, but not in the R201 mutant. Thus, the beta-phosphate may bind in a state-independent fashion to K185 to destabilize channel openings, while R201 interacts with the alpha-phosphate to stabilize a channel closed configuration. Substitution of R192 on the C-terminus and R50 on the N-terminus with different charged residues also affected ATP sensitivity. Based on these results a structural scheme is proposed, which includes features of other recently published models.

Role of adenine nucleotides in insulin secretion from MIN6 pseudoislets.

Insulin secretion from MIN6 cells configured as cell aggregates by culture on a gelatin substrate (pseudoislets) is enhanced compared to that of MIN6 cells grown as monolayers on tissue culture plastic, indicating the importance of beta-cell-to-beta-cell proximity for insulin release. In this study we have shown that glucose induced a biphasic release of insulin from pseudoislets, whereas the amplitude and duration of the responses of equivalent monolayer cells were much reduced. Purinergic aqonists have been implicated in intercellular communication between beta-cells, so we investigated whether adenine nucleotides co-released with insulin are responsible for the enhanced secretory responses of pseudoislets. We have demonstrated that MIN6 cells express purinergic A(1) and P2Y receptors, and that adenine nucleotides increased [Ca(2+)](i) with an efficacy of agonists being ATP > ADP > AMP. However, neither suramin nor the more selective A(1) antagonist 1,3-dipropyl-8-cyclopentylxanthine reduced glucose-induced insulin secretion from pseudoislets, and stimulation of monolayer cells with a range of adenine nucleotides did not enhance glucose-induced secretion. These results suggest that enhanced secretion from MIN6 pseudoislets is not due to increased paracrine/autocrine action of adenine nucleotides.

Adenosine and purinergic mediators of tubuloglomerular feedback.

This review will focus on the role of adenosine and adenine nucleotides as potential mediators of the tubuloglomerular feedback response. The effects of these substances on tubular transport, renin release, and long-term adaptations of tubuloglomerular feedback are worthy of discussion, but will not be considered here.

Diadenosine polyphosphates cause contraction and relaxation in isolated rat resistance arteries.

The effects of diadenosine polyphosphates (APnA; n = 3-6) and adenine nucleotides on contractile reactivity of isolated rat mesenteric resistance arteries (MrA) and superior epigastric arteries (SEA), which display a dense and sparse autonomic innervation, respectively, were evaluated. All agonists examined, except adenosine and AMP, induced contractions. The rank order of potency was similar in both arteries: alpha,beta-methylene ATP (alpha,beta-meATP) > AP5A > AP6A > AP4A > ATP > ADP > AP3A. Contractions were stable during several minutes in SEA but highly transient in MrA. They were reduced after exposure to 10 microM alpha,beta-meATP and by 10 microM of the P2X antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid. During phenylephrine (10 microM)-induced contractions, the agonists induced a further contraction in SEA. In MrA, however, further contraction was followed by marked relaxation. The rank order of relaxing potency was comparable to that of the contractile potency of agonists. Also, the relaxing effects of APnA were blunted by 10 microM pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid and after exposure to alpha,beta-meATP. In vitro and in vivo sympathectomy with 6-hydroxydopamine and removal of the endothelium did not modify the effects of APnA in MrA. Thus, the contractile effects of APnA in resistance arteries 1) are due to a P2X purinoceptor-mediated stimulation of the smooth muscle; 2) depend on the length of the phosphate chain; and 3) are followed by endothelium-independent relaxing effects in MrA but not SEA, which may involve receptors that are similar to those mediating contraction. The regional heterogeneity of APnA effects cannot be attributed to a direct neurogenic influence.

Schizophrenia: a purinergic hypothesis.

Knowledge of the physiological roles of the purinergic system and its influence on other neurotransmitter systems has greatly advanced. In this article, a purinergic model is proposed as an attempt to integrate several findings in schizophrenia. According to this hypothesis, a purinergic system dysfunction would mainly result in reduced adenosinergic activity. This model also addresses the systemic aspects of schizophrenia, based on peripheral roles of purines, such as modulation of the immune system.