ATP Receptor Antagonist Research Articles

Photostimulation of Channelrhodopsin‐2 expressing ventral medullary astrocytes increases sympathetic nerve activity and blood pressure in rats

Astrocytes play an important role in the control of complex central nervous functions by regulating synaptic strength and information processing. Here we explored the potential role of ventral medullary astrocytes in the regulation of central sympathetic tone and blood pressure (BP). Adult rats received bilateral microinjections in the rostral ventrolateral medulla (RVLM) of AVV‐sGFAP‐ChR2(H134R)‐Katushka1.3, an adenovirus that expresses channelrhodopsin‐2 (ChR2) under the control of the astrocyte‐specific promoter GFAP. 7–14 days later, strong ChR2 expression was detected in ventral medullary astrocytes located in close proximity to tyrosine hydroxilase‐immunoreactive RVLM neurons. Unilateral stimulation with blue light (445 nm) increased BP (20.7 mmHg, n=11) and renal nerve sympathetic discharge (SND; 28.7 %, n=11). Application of the ATP receptor antagonist MRS 2179 (200 μM) on the ventral medullary surface decreased baseline SND (15%) and BP (7.5%). Photostimulation after ATP receptor blockade produced signtificantly smaller responses in SND (12.5%) and BP (5%).CONCLUSIONSActivation of ventral medullary astrocytes increases sympathetic activity and blood pressure at least in part via ATP‐dependent mechanisms. This is the first demonstration that astroglia may play an active role in shaping the patterns of central sympathetic outflow.

Optogenetic Analysis of Area‐Specific Glial‐Neuronal Signalling

We studied glia‐neuronal signalling in two brainstem areas, the retrotrapezoid nucleus (RTN) and the locus coeruleus (LC). RTN and LC neurons were visualized using adenoviral expression of DsRed2 under control of PRSx8 promoter. For optogenetic selective stimulation of astrocytes we have generated an adenoviral vector AVV‐sGFAP‐ChR2(H134R)‐Katushka1.3. We first patched AVV‐sGFAP‐ChR2(H134R)‐Katushka1.3‐transduced astrocytes and found that flashing 470 nm light induced precisely timed depolarisations (+18.8 ± 2.0 mV, n=7, p<0.001). We then patched RTN and LC neurons and stimulated adjacent astrocytes using light. This evoked long lasting depolarization of both RTN (+3.57 ± 0.5 mV, n=9, p<0.001) and LC neurons (+ 3.08 ± 0.33 mV, n=14, p<0.001). Responses of the RTN neurons were markedly reduced by the ATP receptor antagonist MRS2179 (10 μm), depolarisation +0.7mV, n=7, p<0.001). In contrast, neither MRS 2179 nor TNP‐ATP (10 μm) had any effect on of the responses in LC neurons (n=6, p=0.71 for MRS2179 and n=9, p=0.86 for TNP‐ATP). At the same time depolarisations in LC neurons were prevented by an NMDA receptor antagonist dAPV (50 μm, n=8, p<0.01) and AMPA receptor antagonist CNQX (10 μm, n=5, p<0.001). Therefore, glia‐neuronal information transfer occurs in area‐ and transmitter‐dependent manner.

Astrocytes Control Breathing Through pH-Dependent Vesicular Release of Atp

Extracellular signalling mediated by ATP has been associated with central chemosensory function. It has been shown that increase in pCO2 in the arterial blood triggers an immediate release of ATP from the chemosensitive regions located on the ventral surface of the medulla oblongata (VMS).Using in vitro and in vivo preparations and novel genetically encoded Ca2+ indicator based on cyclically permutated GFP - Case 12, we show that astrocytes in the ventral regions of the medulla oblongata are highly sensitive to changes in pH. Decrease in extracellular pH from 7.4 to 7.2 induced transient increases in [Ca2+]i in astrocytes from dissociated neuro-glial cultures prepared from the VMS, in ventral astrocytes of organotypic brainstem slice cultures and in acute horizontal brainstem slices of adult rats, as well as on the VMS in anaesthetized and artificially ventilated rats. ATP receptor antagonists such as MRS2179, PPADS and TNP-ATP effectively blocked [Ca2+]i responses evoked by lowering pH in VMS astrocytes. ATP hydrolyzing enzyme apyrase completely prevented propagation of [Ca2+]i excitation in VMS astrocytes evoked by lowering external pH. These data suggest that Ca2+ responses induced by lowering external pH are mediated by ATP release and subsequent activation of P2 receptors. Inhibitors of vesicular transport brefeldine A and bafilomycin A both effectively abolished [Ca2+]i excitation of VMS astrocytes evoked by lowering external pH. Incubation of astrocytes with FM 1-46 dye (vesicular marker) leads to selective labelling of intracellular vesicles. Acidification of external medium induced decrease in the fluorescence intensity of vesicles labelled with FM 1-43. These data suggest that VMS astrocytes respond to a decrease in pH by releasing ATP via vesicular exocytosis.

α-Hemolysin from Escherichia coli uses endogenous amplification through P2X receptor activation to induce hemolysis

Escherichia coli is the dominant facultative bacterium in the normal intestinal flora. E. coli is, however, also responsible for the majority of serious extraintestinal infections. There are distinct serotypical differences between facultative and invasive E. coli strains. Invasive strains frequently produce virulence factors such as alpha-hemolysin (HlyA), which causes hemolysis by forming pores in the erythrocyte membrane. The present study reveals that this pore formation triggers purinergic receptor activation to mediate the full hemolytic action. Non-selective ATP-receptor (P2) antagonists (PPADS, suramin) and ATP scavengers (apyrase, hexokinase) concentration dependently inhibited HlyA-induced lysis of equine, murine, and human erythrocytes. The pattern of responsiveness to more selective P2-antagonists implies that both P2X(1) and P2X(7) receptors are involved in HlyA-induced hemolysis in all three species. In addition, our results also propose a role for the pore protein pannexin1 in HlyA-induced hemolysis, as non-selective inhibitors of this channel significantly reduced hemolysis in the three species. In conclusion, activation of P2X receptors and possibly also pannexins augment hemolysis induced by the bacterial toxin, HlyA. These findings potentially have clinical perspectives as P2 antagonists may ameliorate symptoms during sepsis with hemolytic bacteria.

Astrocytic Ca2+ Waves Guide CNS Growth Cones to Remote Regions of Neuronal Activity

Activity plays a critical role in network formation during developmental, experience-dependent, and injury related remodeling. Here we report a mechanism by which axon trajectory can be altered in response to remote neuronal activity. Using photoconductive stimulation to trigger high frequency action potentials in rat hippocampal neurons in vitro, we find that activity functions as an attractive cue for growth cones in the local environment. The underlying guidance mechanism involves astrocyte Ca2+ waves, as the connexin-43 antagonist carbenoxolone abolishes the attraction when activity is initiated at a distance greater than 120 µm. The asymmetric growth cone filopodia extension that precedes turning can be blocked with CNQX (10 µM), but not with the ATP and adenosine receptor antagonists suramin (100 µM) and alloxazine (4 µM), suggesting non-NMDA glutamate receptors on the growth cone mediate the interaction with astrocytes. These results define a potential long-range signalling pathway for activity-dependent axon guidance in which growth cones turn towards directional, temporally coordinated astrocyte Ca2+ waves that are triggered by neuronal activity. To assess the viability of the guidance effect in an injury paradigm, we performed the assay in the presence of conditioned media from lipopolysaccharide (LPS) activated purified microglial cultures, as well as directly activating the glia present in our co-cultures. Growth cone attraction was not inhibited under these conditions, suggesting this mechanism could be used to guide regeneration following axonal injury.

P2X7Receptor Activation Amplifies Lipopolysaccharide-Induced Vascular Hyporeactivity via Interleukin-1β Release

Lipopolysaccharide (LPS) stimulates cytoplasmic accumulation of pro-interleukin (IL)-1beta. Activation of P2X(7) receptors stimulates conversion of pro-IL-1beta into mature IL-1beta, which is then secreted. Because both LPS (in vivo) and IL-1beta (in vitro) decrease vascular reactivity to contractile agents, we hypothesized the following: 1) P2X(7) receptor activation contributes to LPS-induced vascular hyporeactivity, and 2) IL-1beta mediates this change. Thoracic aortas were obtained from 12-week-old male C57BL/6 mice. The aortic rings were incubated for 24 h in Dulbecco's modified Eagle's medium, LPS, benzoylbenzoyl-ATP (BzATP; P2X(7) receptor agonist), LPS plus BzATP, oxidized ATP (oATP; P2X(7) receptor antagonist), or oATP plus LPS plus BzATP. After the treatment, the rings were either mounted in a myograph for evaluation of contractile activity or homogenized for IL-1beta and inducible nitric-oxide synthase (iNOS) protein measurement. In endothelium-intact aortic rings, phenylephrine (PE)-induced contractions were not altered by incubation with LPS or BzATP, but they significantly decreased in aortic rings incubated with LPS plus BzATP. Treatment with oATP or IL-1ra (IL-1beta receptor antagonist) reversed LPS plus BzATP-induced hyporeactivity to PE. In the presence of N(G)-nitro-l-arginine methyl ester or N-([3-(aminomethyl)phenyl]methyl)ethanimidamide (selective iNOS inhibitor), the vascular hyporeactivity induced by LPS plus BzATP on PE responses was not observed. BzATP augmented LPS-induced IL-1beta release and iNOS protein expression, and these effects were also inhibited by oATP. Moreover, incubation of endothelium-intact aortic rings with IL-1beta induced iNOS protein expression. Thus, activation of P2X(7) receptor amplifies LPS-induced hyporeactivity in mouse endothelium-intact aorta, which is associated with IL-1beta-mediated release of nitric oxide by iNOS.

Phrenic long‐term facilitation is induced by acute intermittent hypoxia despite systemic ATP receptor antagonist attenuation of hypoxic phrenic response in rats

Phrenic long‐term facilitation (pLTF) is a form of respiratory plasticity induced by acute intermittent hypoxia (AIH; Mitchell et al., 2001). Although pLTF is elicited in carotid denervated rats, its magnitude is reduced in proportion to the reduction of the hypoxic phrenic response (~35% normal; Bavis and Mitchell, 2002). Thus, the role of residual chemosensory afferent activity and hypoxic phrenic responses in pLTF induction remains unclear. Since ATP is necessary for hypoxic stimulation of carotid chemoafferent activity and the hypoxic ventilatory response (Rong et al., 2003), we tested the hypothesis that systemic P2X receptor antagonism would block both the hypoxic phrenic response and pLTF following AIH in anesthetized rats. Pyridoxal‐phosphate‐6‐azophenyl‐2',4'‐disulfonic acid (PPADS; 100 mg/kg IV), a non‐selective P2X receptor antagonist, was administered to anesthetized, vagotomized, paralyzed and ventilated male Sprague‐Dawley rats prior to AIH (3, 5‐min episodes of 10% O2; 5 min intervals). Although PPADS strongly attenuated the hypoxic phrenic amplitude response (3 ± 5% baseline), significant pLTF was observed in phrenic burst amplitude 60 min post‐AIH (34±8% baseline, n=4; p < 0.05). Thus, AIH initiates mechanisms of pLTF independently from the hypoxic phrenic response and, presumably, chemoafferent neuron activation.Supported by: NIH HL80209 and T32RR17503

Activation of Urothelial Transient Receptor Potential Vanilloid 4 by 4α-Phorbol 12,13-Didecanoate Contributes to Altered Bladder Reflexes in the Rat

The ion channel transient receptor potential vanilloid (TRPV) 4 can be activated by hypo-osmolarity, heat, or certain lipid compounds. Here, we demonstrate expression of functional TRPV4 protein in the urothelium lining the renal pelvis, ureters, urinary bladder, and urethra. Exposure of cultured rat urothelial cells from the urinary bladder to the TRPV4-selective agonist 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) promoted Ca2+ influx, evoked ATP release, and augmented the ATP release evoked by hypo-osmolarity. In awake rats during continuous infusion cystometrograms, intravesical administration of 4alpha-PDD (10-100 microM) increased maximal micturition pressure by 51%, specifically by augmenting the portion of each intravesical pressure wave that follows high-frequency urethral oscillations and voiding. This unusual pharmacological effect was prevented by intravesical pretreatment with the nonselective ATP receptor antagonist, pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (100 microM), systemic treatment with the selective P2X3 purinergic antagonist 5-([(3-phenoxybenzyl)[1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]carbonyl)-1,2,4-benzenetricarboxylic acid (A317491) (250 micromol/kg), or urethane anesthesia, but was unaffected by capsaicin pretreatment (100 mg/kg s.c.) or denervation of the urethral sphincter. 4Alpha-PDD (1-100 microM) did not alter the contractility to electrical stimulation of excised bladder strips. We conclude that activation of urothelial TRPV4 by 4alpha-PDD and release of mediators such as ATP trigger a novel neural mechanism that regulates the late phase of detrusor muscle contraction after micturition. These data raise the possibility that TRPV4 channels in the urothelium could contribute to abnormal bladder activity.

ATP MODULATES THE RELEASE OF NORADRENALINE THROUGH TWO DIFFERENT PREJUNCTIONAL RECEPTORS ON THE ADRENERGIC NERVES OF RAT PROSTATE

1. The effects of adenosine and ATP receptor agonists on the release of endogenous noradrenaline from electrically stimulated (2 Hz, 0.1 msec) rat prostate were examined in order to clarify the pharmacological properties of prejunctional receptors for adenosine and ATP on the adrenergic nerve varicosities in the prostate. Noradrenaline was quantified by HPLC coupled with electrochemical detection techniques. 2. Both adenosine and ATP receptor agonists (1 micromol/L) inhibited noradrenaline release and the relative order of inhibitory effect was N(6)-cyclopentyl-adenosine (CPA) > 5'-N-ethylcarboxamidoadenosine > 2-chloroadenosine > adenosine > 2-methylthio-ATP (2mSATP) > AMP > ATP. 3. The adenosine receptor agonist CPA (1 nmol/L-1 micromol/L) and the ATP receptor agonist 2mSATP (100 nmol/L-100 micromol/L) inhibited the stimulation-induced release of noradrenaline in a concentration-dependent manner. The concentrations of CPA and 2mSATP that produced 50% inhibition of noradrenaline release were 9.6 nmol/L and 1.4 micromol/L, respectively. 4. 1,3-Dipropyl-8-cyclopentylxanthine, an adenosine A(1) receptor antagonist, significantly reduced the inhibitory effects of not only CPA, but also 2mSATP. 5. Suramin, an ATP receptor antagonist, significantly reduced the inhibitory effects of 2mSATP, but not those of CPA. 6. Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid, another ATP receptor antagonist, had no effect on the inhibitory action of either agonist. 7. These results suggest that, in the sympathetic nerve terminals of rat prostate, adenosine and ATP induce inhibition of noradrenaline release via the activation of adenosine A(1) and/or xanthine-sensitive ATP receptors, which play an inhibitory regulatory role in adrenergic neurotransmission in the prostate.

Cosignaling of adenosine and adenosine triphosphate in hypobaric hypoxia-induced hypothermia

Purines, that is, adenosine and ATP, are not only products of metabolism but are also neurotransmitters. Indeed, purinergic neurotransmission is involved in thermoregulatory processes that occur during normoxia. Exposure to severe hypoxia elicits a sharp decrease in body core temperature (T(CO)), and adenosinergic mechanisms have been suspected to be responsible for this hypothermia. Because ATP per se and its metabolite adenosine could have complex interactions in some neural networks, we hypothesize that both adenosine and ATP are involved in the central mechanism of hypoxia-induced hypothermia. Their role in the thermoregulatory process was therefore investigated in a 24-h hypobaric hypoxia (Fi(O2) = 10%), using CGS-15943, a nonselective antagonist of adenosine receptors, and suramin, an ATP receptor antagonist. T(CO) and spontaneous activity (A(S)) were monitored by telemetry in conscious rats, receiving CGS-15943 (10 mg/kg ip), suramin (7 nmol icv), or both. The same treatments were done in normoxia to evaluate the specificity of their thermoregulatory action observed in hypoxia. Suramin/CGS-15943 treatment blunted the profound hypothermia observed in control rats throughout the hypoxia exposure, whereas CGS-15943 treatment blunted hypothermia during only 3 h, and suramin treatment had no effect. These results suggest that suramin potentiates the CGS-15943 effects and consequently that adenosine and ATP signaling act in synergy. In normoxia, suramin/CGS-15943 induced an increase in T(CO) but to a far lesser extent than observed in hypoxia. Thus it might be suggested that the suramin/CGS-15943 blunting of hypoxia-induced hypothermia would be specific to hypoxia-induced mechanisms.

ATP-γ-S shifts the operating point of outer hair cell transduction towards scala tympani

ATP receptor agonists and antagonists alter cochlear mechanics as measured by changes in distortion product otoacoustic emissions (DPOAE). Some of the effects on DPOAEs are consistent with the hypothesis that ATP affects mechano-electrical transduction and the operating point of the outer hair cells (OHCs). This hypothesis was tested by monitoring the effect of ATP-γ-S on the operating point of the OHCs. Guinea pigs anesthetized with urethane and with sectioned middle ear muscles were used. The cochlear microphonic (CM) was recorded differentially (scala vestibuli referenced to scala tympani) across the basal turn before and after perfusion (20 min) of the perilymph compartment with artificial perilymph (AP) and ATP-γ-S dissolved in AP. The operating point was derived from the cochlear microphonics (CM) recorded in response low frequency (200 Hz) tones at high level (106, 112 and 118 dB SPL). The analysis procedure used a Boltzmann function to simulate the CM waveform and the Boltzmann parameters were adjusted to best-fit the calculated waveform to the CM. Compared to the initial perfusion with AP, ATP-γ-S (333 μM) enhanced peak clipping of the positive peak of the CM (that occurs during organ of Corti displacements towards scala tympani), which was in keeping with ATP-induced displacement of the transducer towards scala tympani. CM waveform analysis quantified the degree of displacement and showed that the changes were consistent with the stimulus being centered on a different region of the transducer curve. The change of operating point meant that the stimulus was applied to a region of the transducer curve where there was greater saturation of the output on excursions towards scala tympani and less saturation towards scala vestibuli. A significant degree of recovery of the operating point was observed after washing with AP. Dose response curves generated by perfusing ATP-γ-S (333 μM) in a cumulative manner yielded an EC 50 of 19.8 μM. The ATP antagonist PPADS (0.1 mM) failed to block the effect of ATP-γ-S on operating point, suggesting the response was due to activation of metabotropic and not ionotropic ATP receptors. Multiple perfusions of AP had no significant effect (118 and 112 dB) or moved the operating point slightly (106 dB) in the direction opposite of ATP-γ-S. Results are consistent with an ATP-γ-S induced transducer change comparable to a static movement of the organ of Corti or reticular lamina towards scala tympani.

Ex vivo evaluation of erythrocytosis-enhanced platelet thrombus formation using the cone and plate(let) analyzer: effect of platelet antagonists.

Red blood cells (RBC) contribute significantly to haemostasis and thrombosis under oscillatory flow conditions, and erythrocytosis has been associated with increased thrombotic risk. To measure the dynamic influences of RBC on platelets, we used a recently described cone and plate(let) analyzer (CPA), evaluating the effect of haematocrit (Hct) on platelet function in whole blood under arterial flow conditions (1800/s, 2 min, 25 degrees C). Anticoagulated blood, reconstituted to varying haematocrits with autologous RBC, demonstrated a significant increase in adherent platelet aggregate formation at Hct levels >45%. This increase was not affected by pretreatment of blood with 0.05 mmol/l aspirin, but was prevented by antagonists of P2Y1, P2Y12, or P2X1, ADP and ATP receptors, and by converting exogenous ADP to ATP with creatine phosphate/creatine phosphokinase. As negligible platelet granule secretion was measured during CPA analysis, but metabolic inhibition of RBC with sodium azide or glutaraldehyde fixation inhibited erythrocytosis-enhanced increases in platelet aggregate size, adenine nucleotides contributing to shear-induced platelet aggregate formation appear to be derived from erythrocytes. These findings support the use of CPA for ex vivo evaluation of the contribution of RBC to platelet function and its inhibition under physiological shear conditions.

Viral dsRNA activates mucin transcription in airway epithelial cells

Double-stranded (ds) RNA is a biologically active component of many viruses including rhinoviruses infecting the upper respiratory tract. Mucus production is a common symptom of such infections. Here, we show that mucin, the glycoprotein subunit of mucus gels, is transcriptionally upregulated in an NF-κB- and p38-dependent manner when homogeneous cultures of epithelial cells are exposed to dsRNA. Furthermore, upstream of p38 in this system, dsRNA stimulates the extracellular release of ATP and activation of cell surface ATP receptors, which are G protein-coupled. This results in the stimulation of phospholipase C and protein kinase C. These findings suggest that ATP receptor antagonists could be used to modulate mucus production induced by virus.

Presence of diadenosine polyphosphates in the aqueous humor: their effect on intraocular pressure.

Adenine dinucleotides are present in many biological systems and may serve as physiological regulators of processes such as neurotransmitter release, vascular tone or corneal hydration. The presence of diadenosine polyphosphates was investigated in New Zealand White rabbit aqueous humor. Diadenosine tetraphosphate (Ap4A) and diadenosine pentaphosphate (Ap5A) were identified and quantified in the aqueous humor with concentrations of 0.34 +/- 0.1 and 0.08 +/- 0.01 microM, respectively. The effects of topical corneal application of diadenosine pyrophosphate (Ap2A), diadenosine triphosphate (Ap3A), Ap4A, and Ap5A on intraocular pressure in rabbits were also studied. Ap2A, Ap3A, and Ap5A increased intraocular pressure with threshold doses of approximately 0.1 to 1.0 micro g. 10 microl(-1). Ap4A decreased intraocular pressure with an IC50 value of 0.12 micro g. 10 microl(-1) (or 0.13 nmol). Cross-desensitization studies suggested the activation of a P2X receptor for the hypotensive effect of Ap4A and a P2Y receptor in the case of Ap5A. The ATP receptor antagonists (all 100 micro g. 10 microl(-1)), pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), suramin, and reactive blue 2 (RB-2) alone had no effect on intraocular pressure but attenuated responses to diadenosine polyphosphates by approximately 80%. It is concluded that Ap2A, Ap3A, and Ap5A increase intraocular pressure, and Ap4A decreases intraocular pressure via mechanisms that involve P2 receptors, and that Ap4A present in aqueous humor may serve to regulate intraocular pressure. Furthermore, we suggest that topical application of Ap4A to the cornea has therapeutic potential for lowering intraocular pressure, a major risk factor for glaucoma.

ATP-induced calcium oscillations and change of P2Y subtypes with culture conditions in HeLa cells.

ATP, UTP, ADP and UDP induced intracellular Ca(2+) responses and oscillations in HeLa cells that sometimes lasted over 1 h. The response is due to the activation of P2Ys, G-protein coupled ATP receptors, because the oscillations persisted for several minutes even in Ca(2+)-free solution, and suramin and PPADS, antagonists of ATP receptors, partially inhibited the response. The potency of these nucleotides varied with the culture or cell conditions, i.e. UTP was generally most potent but in some cases UDP was more potent; responses to UDP were variable while those to ATP were constant. In addition, Ca(2+) responses to ATP and UDP were additive. These findings suggested the existence of two or more subtypes of P2Ys in HeLa cells. RT-PCR experiments revealed the existence of P2Y(2), P2Y(4) and P2Y(6). Recovery from starvation (culture in FBS-free medium overnight and re-addition of FBS) increased the responses to UTP and UDP but not to ATP, suggesting that the number or activity of P2Y(6) and/or P2Y(4) receptors may increase with cell proliferation in HeLa cells.

A Role for Purinergic Receptors at the Inner Hair Cell-Afferent Synapse?

Previously published evidence is reviewed for a functional role of ATP and its receptors at the chemical synapse between the mammalian inner hair cell and the primary afferent dendrites of the VIIIth nerve. New findings are also presented, both from gross cochlear potentials and single neurone recordings during intracochlear perfusion of ATP-receptor antagonists and agonists. Both the previous and present results are consistent with the notion that endogenous ATP may act to regulate the excitability of the primary afferent dendrite, possibly acting on P2X₂ receptors.

Promotion of formation of amyloid fibrils by aluminium adenosine triphosphate (AlATP).

The formation of amyloid fibrils is considered to be an important step in the aetiology of Alzheimer's disease and other amyloidoses. Fibril formation in vitro has been shown to depend on many different factors including modifications to the amino acid profile of fibrillogenic peptides and interactions with both large and small molecules of physiological significance. How these factors might contribute to amyloid fibril formation in vivo is not clear as very little is known about the promotion of fibril formation in undersaturated solutions of amyloidogenic peptides. We have used thioflavin T fluorescence and reverse phase high performance liquid chromatography to show that ATP, and in particular AlATP, promoted the formation of thioflavin T-reactive fibrils of beta amyloid and, an unrelated amyloidogenic peptide, amylin. Evidence is presented that induction of fibril formation followed the complexation of AIATP by one or more monomers of the respective peptide. However, the complex formed could not be identified directly and it is suggested that AlATP might be acting as a chaperone in the assembly of amyloid fibrils. The effect of AlATP was not mimicked by either AlADP or AlAMP. However, it was blocked by suramin, a P2 ATP receptor antagonist, and this has prompted us to speculate that the precursor proteins to beta amyloid and amylin may be substrates or receptors for ATP in vivo.

Reactive blue 2 induces calcium oscillations in HeLa cells.

Reactive Blue 2 (RB), which is used as an ATP receptor antagonist, induced Ca(2+) oscillations in HeLa cells. RB-induced Ca(2+) oscillations were abolished in Ca(2+)-free solution. RB, however, did not affect Ca(2+) influx measured by Mn(2+) quenching. The PLC cascade and intracellular Ca(2+) release were involved as U73122 and thapsigargin inhibited RB-induced Ca(2+) oscillations. RB enhanced a Ca(2+) response to histamine that is linked to the PLC cascade. RB may activate the PLC cascade in an extracellular Ca(2+)-dependent manner and induce Ca(2+) oscillations.

Glucose deprivation and chemical hypoxia: neuroprotection by P2 receptor antagonists

In this work we investigate cell survival after glucose deprivation and/or chemical hypoxia and we analyse the neuroprotective properties of selected antagonists of P2 ATP receptors. We find that in rat cerebellar granule neurones, the antagonist basilen blue prevents neuronal death under hypoglycaemia. Basilen blue acts through a wide temporal range and it retains its efficacy under chemically induced hypoxic conditions, in the presence of the respiratory inhibitors of mitochondria electron transport chain complexes II (3-nitropropionic acid) and III (antimycin A). In spite of the presence of these compounds, basilen blue maintains normal intracellular ATP levels. It furthermore prevents neuronal death caused by agents blocking the mitochondrial calcium uptake (ruthenium red) or discharging the mitochondrial membrane potential (carbonyl cyanide m-chlorophenylhydrazone). Inhibition of poly (ADP-ribose) polymerase, modulation of the enzyme GAPDH and mitochondrial transport of mono-carboxylic acids are not conceivable targets for the action of basilen blue. Survival is sustained by basilen blue also in CNS primary cultures from hippocampus and in PNS sympathetic-like neurones. Partial neuroprotection is furthermore provided by three additional P2 receptor antagonists: suramin, pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid 4-sodium and 4,4′-diisothiocyanatostilbene-2,2′disulphonic acid. Our data suggest the exploitation of selected P2 receptor antagonists as potential neuroprotective agents.

Hypoglycaemia-induced cell death: features of neuroprotection by the P2 receptor antagonist basilen blue

Our previous work in neuronal cultures has shown that several antagonists of P2 ATP receptors prevent cell death evoked by hypoglycaemia, chemical hypoxia, mitochondria dysfunction, as well as glutamate-dependent excitotoxicity and low potassium-induced apoptosis. Experiments are now designed to examine which biological pathway contributes to cell death/survival under glucose starvation. We show here that, consequently to hypoglycaemic insults, cerebellar granule neurones undergo a combination of apoptosis and necrosis both inhibited by the P2 receptor antagonist basilen blue. This is demonstrated by morphological and biochemical features, such as TdT-mediated dUTP-biotin nick end-labelling, fluorescent staining of nuclear chromatin using Hoechst 33258, direct counting of intact viable nuclei and extracellular releasing of the cytosolic enzyme LDH. Furthermore, we show that hypoglycaemia induces outflow of cytochrome c from mitochondria and it up-regulates heat-shock proteins HSP70, but not HSP90, glucose-regulated proteins GRP75 and GRP78, as well as expression and activity of the enzyme caspase-2. Basilen blue can modulate only some of these effects. Our data contribute to dissect the role played by P2 receptor antagonism in sustaining neuroprotection against metabolic stresses.