Autocrine signaling through ATP release represents a novel mechanism for cell volume regulation.

Recovery of cell volume in response to osmotic stress is mediated in part by increases in the Cl- permeability of the plasma membrane. These studies evaluate the hypothesis that ATP release and autocrine stimulation of purinergic (P2) receptors couple increases in cell volume to opening of Cl- channels. In HTC rat hepatoma cells, swelling induced by hypotonic exposure increased membrane Cl- current density to 44.8 +/- 7.1 pA/pF at -80 mV. Both the rate of volume recovery and the increase in Cl- permeability were inhibited in the presence of the ATP hydrolase apyrase (3 units/ml) or by exposure to the P2 receptor blockers suramin and Reactive Blue 2 (10-100 microM). Cell swelling also stimulated release of ATP. Hypotonic exposure increased the concentration of ATP in the effluent of perfused cells by 170 +/- 36 nM in the presence of a nucleotidase inhibitor (P < 0.01). In whole-cell recordings with ATP as the charge carrier, cell swelling increased membrane current density approximately 30-fold to 16.5 +/- 10.4 pA/pF. These findings indicate that increases in cell volume lead to efflux of ATP through opening of a conductive pathway consistent with a channel, and that extracellular ATP is required for recovery from swelling. ATP may function as an autocrine factor that couples increases in cell volume to opening of Cl- channels through stimulation of P2 receptors.

P2X4: an ATP-activated ionotropic receptor cloned from rat brain.

Extracellular ATP exerts pronounced biological actions in virtually every organ or tissue that has been studied. In the central and peripheral nervous system, ATP acts as a fast excitatory transmitter in certain synaptic pathways [Evans, R.J., Derkach, V. & Surprenant, A. (1992) Nature (London) 357, 503-505; Edwards, F.A., Gigg, A.J. & Colquhoun, D. (1992) Nature (London) 359, 144-147]. Here, we report the cloning and characterization of complementary DNA from rat brain, encoding an additional member (P2X4) of the emerging multigenic family of ligand-gated ATP channels, the P2X receptors. Expression in Xenopus oocytes gives an ATP-activated cation-selective channel that is highly permeable to Ca2+ and whose sensitivity is modulated by extracellular Zn2+. Surprisingly, the current elicited by ATP is almost insensitive to the common P2X antagonist suramin. In situ hybridization reveals the expression of P2X4 mRNA in central nervous system neurons. Northern blot and reverse transcription-PCR (RT-PCR) analysis demonstrate a wide distribution of P2X4 transcripts in various tissues, including blood vessels and leukocytes. This suggests that the P2X4 receptor might mediate not only ATP-dependent synaptic transmission in the central nervous system but also a wide repertoire of biological responses in diverse tissues.

Mouse Leydig cells express multiple P2X receptor subunits

ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X(1)-P2X(7)) and seven heteromeric receptors (P2X(1/2), P2X(1/4), P2X(1/5), P2X(2/3), P2X(2/6), P2X(4/6), P2X(4/7)) have been described. ATP treatment of Leydig cells leads to an increase in [Ca(2+)](i) and testosterone secretion, supporting the hypothesis that Ca(2+) signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X(2). In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X(2), P2X(4), P2X(6), and P2X(7) subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 mu M ivermectin induced an increase (131.2 +/- 5.9%) and 3 mu M ivermectin a decrease (64.2 +/- 4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X(4) subunits. P2X(7) receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel, possibly P2X(2/4/6), is present in Leydig cells, but with an electrophysiological and pharmacological phenotype characteristic of the P2X(2) subunit.

Dissecting cross-talk between microglia and motoneurons in ALS: signaling events and soluble factors

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Procedures to characterize and study P2Z/P2X7 purinoceptor: flow cytometry as a promising practical, reliable tool

The expression of P2Z/P2X7 purinoceptor in different cell types is well established. This receptor is a member of the ionotropic P2X receptor family, which is composed by seven cloned receptor subtypes (P2X1 - P2X7). Interestingly, the P2Z/P2X7 has a unique feature of being linked to a non-selective pore which allows the passage of molecules up to 900 Da depending on the cell type. Early studies of P2Z/P2X7 purinoceptor were exclusively based on classical pharmacological studies but the recent tools of molecular biology have enriched the analysis of the receptor expression. The majority of assays and techniques chosen so far to study the expression of P2Z/P2X7 receptor explore directly or indirectly the effects of the opening of P2Z/P2X7 linked pore. In this review we describe the main techniques used to study the expression and functionality of P2Z/P2X7 receptor. Additionally, the increasing need and importance of a multifunctional analysis of P2Z/P2X7 expression based on flow cytometry technology is discussed, as well as the adoption of a more complete analysis of P2Z/P2X7 expression involving different techniques.

Analysis of the NTPDase and ecto-5'-nucleotidase profiles in serum-limited Trichomonas vaginalis

Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) family members, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10% serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1% serum). The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi) released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.

Iron from haemoglobin and haemin modulates nucleotide hydrolysis inTrichomonas vaginalis

Extracellular ATP may act as a danger signalling molecule, inducing inflammation and immune responses in infection sites. The ectonucleotidases NTPDase and ecto-5’-nucleotidase are enzymes that modulate extracellular nucleotide levels; these enzymes have been previously characterised in Trichomonas vaginalis. Iron plays an important role in the complex trichomonal pathogenesis. Herein, the effects of iron on growth, nucleotide hydrolysis and NTPDase gene expression in T. vaginalisisolates from female and male patients were evaluated. Iron from different sources sustainedT. vaginalis growth. Importantly, iron from haemoglobin (HB) and haemin (HM) enhanced NTPDase activity in isolates from female patients and conversely reduced the enzyme activity in isolates from male patients. Iron treatments could not alter the NTPDase transcript levels in T. vaginalis. Furthermore, our results reveal a distinct ATP, ADP and AMP hydrolysis profile between isolates from female and male patients influenced by iron from HB and HM. Our data indicate the participation of NTPDase and ecto-5’-nucleotidase in the establishment of trichomonas infection through ATP degradation and adenosine production influenced by iron.

PDMP blocks brefeldin a-induced retrograde membrane transport from Golgi to ER: evidence for involvement of calcium homeostasis and dissociation from sphingolipid metabolism

In this study, we show that an inhibitor of sphingolipid biosynthesis, d,l-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when BFA was allowed to exert its effect before PDMP addition, PDMP could not ¿rescue¿ the Golgi compartment. Evidence is presented showing that this action of PDMP is indirect, which means that the direct target is not sphingolipid metabolism at the Golgi apparatus. A fluorescent analogue of PDMP, 6-(N-[7-nitro-2,1,3-benzoxadiazol-4-yl]amino)hexanoyl-PDMP (C6-NBD-PDMP), did not localize in the Golgi apparatus. Moreover, the effect of PDMP on membrane flow did not correlate with impaired C6-NBD-sphingomyelin biosynthesis and was not mimicked by exogenous C6-ceramide addition or counteracted by exogenous C6-glucosylceramide addition. On the other hand, the PDMP effect was mimicked by the multidrug resistance protein inhibitor MK571. The effect of PDMP on membrane transport correlated with modulation of calcium homeostasis, which occurred in a similar concentration range. PDMP released calcium from at least two independent calcium stores and blocked calcium influx induced by either extracellular ATP or thapsigargin. Thus, the biological effects of PDMP revealed a relation between three important physiological processes of multidrug resistance, calcium homeostasis, and membrane flow in the ER/ Golgi system.

Regulation and glucocorticoid-independent induction of lipocortin I in cultured astrocytes.

Stimulation of prostaglandin (PG) release in rat astroglial cultures by various substances, including phorbol esters, melittin, or extracellular ATP, has been reported recently. It is shown here that glucocorticoids (GCs) reduced both basal and stimulated PGD2 release. Hydrocortisone, however, did not inhibit ATP-, calcium ionophore A23187-, or tetradecanoyl phorbol acetate (TPA)-stimulated arachidonic acid release, and only TPA stimulations were affected by dexamethasone. GC-mediated inhibition of PGD2 release thus appeared to exclude regulation at the phospholipase A2 (PLA2) level. Therefore, the effects of GCs on the synthesis of lipocortin I (LC I), a potent, physiological inhibitor of PLA2, were studied in more detail. Dexamethasone was not able to enhance de novo synthesis of LC I in freshly seeded cultures and failed to increase LC I synthesis in 2-3-week-old cultures. It is surprising that LC I was the major LC synthesized in those cultures, and marked amounts accumulated with culture time, reaching plateau levels at approximately day 10. In contrast, LC I was barely detectable in vivo. This tonic inhibition of PLA2 is the most likely explanation for unsuccessful attempts to evoke PG release in astrocyte cultures by various physiological stimuli. GC receptor antagonists (progesterone and RU 38486) given throughout culture time reduced LC I accumulation and simultaneously increased PGD2 release. Nonetheless, a substantial production of LC I persisted in the presence of antagonists. Therefore, LC I induction did not seem to involve GC receptor activation. This was confirmed in serum- and GC-free brain cell aggregate cultures. Here also a marked accumulation of LC I was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

An acid-sensing ion channel that detects ischemic pain

Ischemic pain occurs when there is insufficient blood flow for the metabolic needs of an organ. The pain of a heart attack is the prototypical example. Multiple compounds released from ischemic muscle likely contribute to this pain by acting on sensory neurons that innervate muscle. One such compound is lactic acid. Here, we show that ASIC3 (acid-sensing ion channel #3) has the appropriate expression pattern and physical properties to be the detector of this lactic acid. In rats, it is expressed only in sensory neurons and then only on a minority (~40%) of these. Nevertheless, it is expressed at extremely high levels on virtually all dorsal root ganglion sensory neurons that innervate the heart. It is extraordinarily sensitive to protons (Hill slope 4, half-activating pH 6.7), allowing it to readily respond to the small changes in extracellular pH (from 7.4 to 7.0) that occur during muscle ischemia. Moreover, both extracellular lactate and extracellular ATP increase the sensitivity of ASIC3 to protons. This final property makes ASIC3 a "coincidence detector" of three molecules that appear during ischemia, thereby allowing it to better detect acidosis caused by ischemia than other forms of systemic acidosis such as hypercapnia.

Gadolinium increases the vascular reactivity of rat aortic rings

Gadolinium (Gd) blocks intra- and extracellular ATP hydrolysis. We determined whether Gd affects vascular reactivity to contractile responses to phenylephrine (PHE) by blocking aortic ectonucleoside triphosphate diphosphohydrolase (E-NTPDase). Wistar rats of both sexes (260-300 g, 23 females, 7 males) were used. Experiments were performed before and after incubation of aortic rings with 3 µM Gd. Concentration-response curves to PHE (0.1 nM to 0.1 mM) were obtained in the presence and absence of endothelium, after incubation with 100 µM L-NAME, 10 µM losartan, or 10 µM enalaprilat. Gd significantly increased the maximum response (control: 72.3 ± 3.5; Gd: 101.3 ± 6.4%) and sensitivity (control: 6.6 ± 0.1; Gd: 10.5 ± 2.8%) to PHE. To investigate the blockade of E-NTDase activity by Gd, we added 1 mM ATP to the bath. ATP reduced smooth muscle tension and Gd increased its relaxing effect (control: -33.5 ± 4.1; Gd: -47.4 ± 4.1%). Endothelial damage abolished the effect of Gd on the contractile responses to PHE (control: 132.6 ± 8.6; Gd: 122.4 ± 7.1%). L-NAME + Gd in the presence of endothelium reduced PHE contractile responses (control/L-NAME: 151.1 ± 28.8; L-NAME + Gd: 67.9 ± 19% AUC). ATP hydrolysis was reduced after Gd administration, which led to ATP accumulation in the nutrient solution and reduced ADP concentration, while adenosine levels remained the same. Incubation with Gd plus losartan and enalaprilat eliminated the pressor effects of Gd. Gd increased vascular reactivity to PHE regardless of the reduction of E-NTPDase activity and adenosine production. Moreover, the increased reactivity to PHE promoted by Gd was endothelium-dependent, reducing NO bioavailability and involving an increased stimulation of angiotensin-converting enzyme and angiotensin II AT1 receptors.

Purinergic signalling is involved in the malaria parasite Plasmodium falciparum invasion to red blood cells

Plasmodium falciparum, the most important etiological agent of human malaria, is endowed with a highly complex cell cycle that is essential for its successful replication within the host. A number of evidence suggest that changes in parasite Ca(2+) levels occur during the intracellular cycle of the parasites and play a role in modulating its functions within the RBC. However, the molecular identification of Plasmodium receptors linked with calcium signalling and the causal relationship between Ca(2+) increases and parasite functions are still largely mysterious. We here describe that increases in P. falciparum Ca(2+) levels, induced by extracellular ATP, modulate parasite invasion. In particular, we show that addition of ATP leads to an increase of cytosolic Ca(2+) in trophozoites and segmented schizonts. Addition of the compounds KN62 and Ip5I on parasites blocked the ATP-induced rise in [Ca(2+)](c). Besides, the compounds or hydrolysis of ATP with apyrase added in culture drastically reduce RBC infection by parasites, suggesting strongly a role of extracellular ATP during RBC invasion. The use of purinoceptor antagonists Ip5I and KN62 in this study suggests the presence of putative purinoceptor in P. falciparum. In conclusion, we have demonstrated that increases in [Ca(2+)](c) in the malarial parasite P. falciparum by ATP leads to the modulation of its invasion of red blood cells.

Alteration of purinergic P2X(4) and P2X(7) receptor expression in rats with temporal-lobe epilepsy induced by pilocarpine

Although ATP and P2X receptor activity have been lately associated with epilepsy, little is known regarding their exact roles in epileptogenesis. Temporal-lobe epilepsy (TLE) in rat was induced by pilocarpine in order to study changes of hippocampal P2X(2), P2X(4) and P2X(7) receptor expression during acute, latent or chronic phases of epilepsy. During acute and chronic phases increased P2X(7) receptor expression was principally observed in glial cells and glutamatergic nerve terminals, suggesting participation of this receptor in the activation of inflammatory and excitotoxic processes during epileptogenesis. No significant alterations of hippocampal P2X(2) and P2X(4) receptor expression was noted during the acute or latent phase when compared to the control group, indicating that these receptors are not directly involved with the initiation of epilepsy. However, the reduction of hippocampal P2X(4) receptor immunostaining in the chronic phase could reflect neuronal toss or decreased GABAergic signaling. (C) 2008 Elsevier B.V. All rights reserved.

Modulation of intercellular communication in macrophages: possible interactions between GAP junctions and P2 receptors

Gap junctions are connexin-formed channels that play an important role in intercellular communication in most cell types. In the immune system, specifically in macrophages, the expression of connexins and the establishment of functional gap junctions are still controversial issues. Macrophages express P2X(7) receptors that, once activated by the binding of extracellular ATP, lead to the opening of transmembrane pores permeable to molecules of up to 900 Da. There is evidence suggesting an interplay between gap junctions and P2 receptors in different cell systems. Thus, we used ATP-sensitive and -insensitive J774.G8 macrophage cell lines to investigate this interplay. To study junctional communication in J774-macrophage-like cells, we assessed cell-to-cell communication by microinjecting Lucifer Yellow. Confluent cultures of ATP-sensitive J774 cells (ATP-s cells) are coupled, whereas ATP-insensitive J774 cells (ATP-i cells), derived by overexposing J774 cells to extracellular ATP until they do not display the phenomenon of ATP-induced permeabilization, are essentially uncoupled. Western-blot and reverse-transcription polymerase chain reaction assays revealed that ATP-s and ATP-i cells express connexin43 (Cx43), whereas only ATP-s cells express the P2X(7) receptor. Accordingly, ATP-i cells did not display any detectable ATP-induced current under whole-cell patch-clamp recordings. Using immunofluorescence microscopy, Cx43 reactivity was found at the cell surface and in regions of cell-cell contact of ATP-s cells, whereas, in ATP-i cells, Cx43 immunoreactivity was only present in cytosolic compartments. Using confocal microscopy, it is shown here that, in ATP-s cells as well as in peritoneal macrophages, Cx43 and P2X(7) receptors are co-localized to the membrane of ATP-s cells and peritoneal macrophages.

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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)