Heike Franke

P2 receptors and neuronal injury

Extracellular adenosine 5′-triphosphate (ATP) was proposed to be an activity-dependent signaling molecule that regulates glia–glia and glia–neuron communications. ATP is a neurotransmitter of its own right and, in addition, a cotransmitter of other classical transmitters such as glutamate or GABA. The effects of ATP are mediated by two receptor families belonging either to the P2X (ligand-gated cationic channels) or P2Y (G protein-coupled receptors) types. P2X receptors are responsible for rapid synaptic responses, whereas P2Y receptors mediate slow synaptic responses and other types of purinergic signaling involved in neuronal damage/regeneration. ATP may act at pre- and postsynaptic sites and therefore, it may participate in the phenomena of long-term potentiation and long-term depression of excitatory synaptic transmission. The release of ATP into the extracellular space, e.g., by exocytosis, membrane transporters, and connexin hemichannels, is a widespread physiological process. However, ATP may also leave cells through their plasma membrane damaged by inflammation, ischemia, and mechanical injury. Functional responses to the activation of multiple P2 receptors were found in neurons and glial cells under normal and pathophysiological conditions. P2 receptor-activation could either be a cause or a consequence of neuronal cell death/glial activation and may be related to detrimental and/or beneficial effects. The present review aims at demonstrating that purinergic mechanisms correlate with the etiopathology of brain insults, especially because of the massive extracellular release of ATP, adenosine, and other neurotransmitters after brain injury. We will focus in this review on the most important P2 receptor-mediated neurodegenerative and neuroprotective processes and their beneficial modulation by possible therapeutic manipulations.

Astrocytic expression of the Alzheimer's disease β-secretase (BACE1) is stimulus-dependent

The beta‐site APP‐cleaving enzyme (BACE1) is a prerequisite for the generation of β‐amyloid peptides, which give rise to cerebrovascular and parenchymal β‐amyloid deposits in the brain of Alzheimers disease patients. BACE1 is neuronally expressed in the brains of humans and experimental animals such as mice and rats. In addition, we have recently shown that BACE1 protein is expressed by reactive astrocytes in close proximity to β‐amyloid plaques in the brains of aged transgenic Tg2576 mice that overexpress human amyloid precursor protein carrying the double mutation K670N‐M671L. To address the question whether astrocytic BACE1 expression is an event specifically triggered by β‐amyloid plaques or whether glial cell activation by other mechanisms also induces BACE1 expression, we used six different experimental strategies to activate brain glial cells acutely or chronically. Brain sections were processed for the expression of BACE1 and glial markers by double immunofluorescence labeling and evaluated by confocal laser scanning microscopy. There was no detectable expression of BACE1 protein by activated microglial cells of the ameboid or ramified phenotype in any of the lesion paradigms studied. In contrast, BACE1 expression by reactive astrocytes was evident in chronic but not in acute models of gliosis. Additionally, we observed BACE1‐immunoreactive astrocytes in proximity to β‐amyloid plaques in the brains of aged Tg2576 mice and Alzheimers disease patients. GLIA 41:169–179, 2003.

Inhibition of N-Type Voltage-Activated Calcium Channels in Rat Dorsal Root Ganglion Neurons by P2Y Receptors Is a Possible Mechanism of ADP-Induced Analgesia

Patch-clamp recordings from small-diameter rat dorsal root ganglion (DRG) neurons maintained in culture demonstrated preferential inhibition by ATP of high-voltage-activated, but not low-voltage-activated, Ca2+ currents (ICa). The rank order of agonist potency was UTP > ADP > ATP. ATP depressed the ω-conotoxin GVIA-sensitive N-type current only. Pyridoxal-5-phosphate-6-azophenyl-2′,4′-disulphonic acid (PPADS) and 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate tetraammonium, two P2Y1 receptor antagonists, almost abolished the ATP-induced inhibition. Both patch-clamp recordings and immunocytochemistry coupled with confocal laser microscopy indicated a colocalization of functional P2X3 and P2Y1 receptors on the same DRG neurons. Because the effect of ATP was inhibited by intracellular guanosine 5′-O-(2-thiodiphosphate) or by applying a strongly depolarizing prepulse, P2Y1 receptors appear to block ICa by a pathway involving the βγ subunit of a Gq/11 protein. Less efficient buffering of the intracellular Ca2+ concentration ([Ca2+]i) by reducing the intrapipette EGTA failed to interfere with the ATP effect. Fura-2 microfluorimetry suggested that ATP raised [Ca2+]i by a Gα-mediated release from intracellular pools and simultaneously depressed the high external potassium concentration-induced increase of [Ca2+]i by inhibiting ICa via Gβγ. Adenosine 5′-O-(2-thiodiphosphate) inhibited dorsal root-evoked polysynaptic population EPSPs in the hemisected rat spinal cord and prolonged the nociceptive threshold on intrathecal application in the tail-flick assay. These effects were not antagonized by PPADS. Hence, P2Y receptor activation by ADP, which is generated by enzymatic degradation of ATP, may decrease the release of glutamate from DRG terminals in the spinal cord and thereby partly counterbalance the algogenic effect of ATP.

P2 receptor-mediated proliferative effects on astrocytes in vivo

Astrogliosis in response to injury usually represents up‐regulation of glial fibrillary acidic protein (GFAP), hypertrophy, and proliferation. Following pathological events in brain tissue, purine nucleotides and nucleosides are released into the extracellular space. The (patho)physiological importance and molecular mechanisms of the purinoceptor‐mediated effects are nearly unknown. In the present study, the involvement of extracellular ATP in astrogliotic processes via stimulation of P2 receptors was investigated. The structural analogue, 2‐methylthio ATP (2‐MeSATP) and its antagonists reactive blue 2 and pyridoxal‐phosphate‐6‐azophenyl‐2,4‐disulphonic acid (PPADS) were microinfused in situ into the rat nucleus accumbens. The reaction of astrocytes in the nucleus accumbens was investigated by GFAP‐ and 5‐bromo‐2′‐deoxyuridine (BrdU)‐ immunocytochemistry. Tissue injury due to the microinjection procedure caused astrogliosis, which was increased further by 2‐MeSATP. Up‐regulation of GFAP‐immunoreactivity, hypertrophy of astrocytes, and an increase in the number of GFAP‐positive and of GFAP‐/BrdU‐double labeled cells were observed. Reactive blue 2 and PPADS decreased the consequences of tissue injury on astrocytic proliferation when given alone. In addition, both antagonists counteracted the 2‐MeSATP‐induced astrogliosis, supporting the hypothesis that purine nucleotides are involved in these processes via stimulation of P2 receptors in vivo. GLIA 28:190–200, 1999.

The reaction of astrocytes and neurons in the hippocampus of adult rats during chronic ethanol treatment and correlations to behavioral impairments

Chronic ethanol treatment of Wistar rats to 10% (v/v) ethanol over a period of 4, 12, and 36 weeks produced distinct alterations of the glial fibrillary acidic protein immunoreactivity (GFAP-IR) of dorsal hippocampal astrocytes. Ethanol consumption over a period of 4 weeks caused an increase in the total GFAP-IR of the astrocytes. Down-regulation of the total GFAP-IR was measured in all examined brain regions after 36 weeks of ethanol treatment. Prolonged ethanol treatment induced a significant loss of the total number of hippocampal pyramidal and dentate gyrus granule cells. Regional differences in the vulnerability to the neurotoxic effects of chronic ethanol intake over 36 weeks were found: CA3 > CA1 + CA2 > > CA4 > GD. In agreement with the degree of neuronal cell loss, ethanol-induced behavioral impairments were found. The acquisition of maze performance using a complex elevated labyrinth was deteriorated after 36 weeks of ethanol treatment, suggesting a deficit in learning and memory. These findings illustrate the importance of time-response analysis when determining the structural and functional changes produced by chronic ethanol treatment.

Pathophysiology of astroglial purinergic signalling

Astrocytes are fundamental for central nervous system (CNS) physiology and are the fulcrum of neurological diseases. Astroglial cells control development of the nervous system, regulate synaptogenesis, maturation, maintenance and plasticity of synapses and are central for nervous system homeostasis. Astroglial reactions determine progression and outcome of many neuropathologies and are critical for regeneration and remodelling of neural circuits following trauma, stroke, ischaemia or neurodegenerative disorders. They secrete multiple neurotransmitters and neurohormones to communicate with neurones, microglia and the vascular walls of capillaries. Signalling through release of ATP is the most widespread mean of communication between astrocytes and other types of neural cells. ATP serves as a fast excitatory neurotransmitter and has pronounced long-term (trophic) roles in cell proliferation, growth, and development. During pathology, ATP is released from damaged cells and acts both as a cytotoxic factor and a proinflammatory mediator, being a universal “danger” signal. In this review, we summarise contemporary knowledge on the role of purinergic receptors (P2Rs) in a variety of diseases in relation to changes of astrocytic functions and nucleotide signalling. We have focussed on the role of the ionotropic P2X and metabotropic P2YRs working alone or in concert to modify the release of neurotransmitters, to activate signalling cascades and to change the expression levels of ion channels and protein kinases. All these effects are of great importance for the initiation, progression and maintenance of astrogliosis–the conserved and ubiquitous glial defensive reaction to CNS pathologies. We highlighted specific aspects of reactive astrogliosis, especially with respect to the involvement of the P2X7 and P2Y1R subtypes. Reactive astrogliosis exerts both beneficial and detrimental effects in a context-specific manner determined by distinct molecular signalling cascades. Understanding the role of purinergic signalling in astrocytes is critical to identifying new therapeutic principles to treat acute and chronic neurological diseases.

P2 receptor‐types involved in astrogliosis in vivo

In the nucleus accumbens (NAc) of rats, the involvement of P2X and P2Y receptors in the generation of astrogliosis in vivo, was investigated by local application of their respective ligands. The agonists used had selectivities for P2X1,3 (α,β‐methylene adenosine 5′‐triphosphate; α,β‐meATP), P2Y1,12 (adenosine 5′‐O‐(2‐thiodiphosphate; ADP‐β‐S) and P2Y2,4,6 receptors (uridine 5′‐O‐(3‐thiotriphosphate; UTP‐γ‐S). Pyridoxalphosphate‐6‐azophenyl‐2,4‐disulphonic acid (PPADS) was used as a non‐selective antagonist. The astroglial reaction was studied by means of immunocytochemical double‐labelling with antibodies to glial fibrillary acidic protein (GFAP) and 5‐bromo‐2′‐deoxyuridine (BrdU). The agonist‐induced changes in comparison to the artificial cerebrospinal fluid (aCSF)‐treated control side reveal a strong mitogenic potency of ADP‐β‐S and α,β‐meATP, whereas UTP‐γ‐S was ineffective. The P2 receptor antagonist PPADS decreased the injury‐induced proliferation when given alone and in addition inhibited all agonist effects. The observed morphogenic changes included hypertrophy of astrocytes, elongation of astrocytic processes and up‐regulation of GFAP. A significant increase of both GFAP‐immunoreactivity (IR) and GFA‐protein content (by using Western blotting) was found after microinfusion of α,β‐meATP or ADP‐β‐S. In contrast, UTP‐γ‐S failed to increase the GFAP‐IR. The morphogenic effects were also inhibited by pre‐treatment with PPADS. A double immunofluorescence approach with confocal laser scanning microscopy showed the localisation of P2X3 and P2Y1 receptors on the GFAP‐labelled astrocytes. In conclusion, the data suggest that P2Y (P2Y1 or P2Y12) receptor subtypes are involved in the generation of astrogliosis in the NAc of rats, with a possible minor contribution of P2X receptor subtypes.

Cyclic Mechanical Stretch Induces Cardiomyocyte Orientation and Polarization of the Gap Junction Protein Connexin43

Rationale: Cyclic mechanical stretch (CMS) is an important physiological and pathological factor in the heart. Objective: We examined whether CMS can affect localization of gap junctions with regard to the cell axis. Methods and Results: Neonatal rat cardiomyocytes were cultured (7 days) on flexible 6-well plates. Thereafter, cells were kept static or stimulated with CMS (1 Hz; 0, 10, 20% elongation) for 0, 24, or 48 hours (with or without 10 &mgr;mol/L PD98059, 5 &mgr;mol/L BIM I (bisindolylmaleimide I), 2 &mgr;mol/L H8 [N-(2-methlyamino-ethyl)-5-isoquinoline-sulfonamid], or 0.1 &mgr;mol/L angiotensin II. Additionally, cells were exposed to 24 hours of CMS followed by 24 hours of static recovery. CMS (24 hour, 10%) induced elongation of the cardiomyocytes and orientation 79±8° toward the stretch direction. Moreover, the distribution of connexin (Cx)43 together with N-cadherin changed, so that both proteins were accentuated at the cell poles, whereas in nonstretched cells, they were distributed around the cell without preferential localization. Additional angiotensin II reduced polar Cx43 accentuation. The CMS-induced changes in Cx43 were reversible within 24 hours after end of stretch, and could be completely prevented by the MEK1/2 inhibitor PD98059 but not by BIM I or H8. Moreover, stretch resulted in Cx43 protein and Cx43-mRNA upregulation and in a significant upregulation of the phosphorylated forms of ERK1/2, glycogen synthase kinase 3&bgr; and AKT. Furthermore, CMS resulted in a significant increase of the transcription factors activator protein 1 and CREB (cAMP response element–binding protein) in the nucleus. Conclusions: CMS results in self-organization of cardiomyocytes leading to elongated cells orientated transverse to the stretch axis, enhanced Cx43 expression and Cx43 accentuation at the cell poles. The Cx43-changes seem to depend on the ERK1/2 signaling cascade.

Supersensitivity of P2X7 receptors in cerebrocortical cell cultures after in vitro ischemia

Neuronally enriched primary cerebrocortical cultures were exposed to glucose‐free medium saturated with argon (in vitro ischemia) instead of oxygen (normoxia). Ischemia did not alter P2X7 receptor mRNA, although serum deprivation clearly increased it. Accordingly, P2X7 receptor immunoreactivity (IR) of microtubuline‐associated protein 2 (MAP2)‐IR neurons or of glial fibrillary acidic protein (GFAP)‐IR astrocytes was not affected; serum deprivation augmented the P2X7 receptor IR only in the astrocytic, but not the neuronal cell population. However, ischemia markedly increased the ATP‐ and 2′‐3′‐O‐(4‐benzoylbenzoyl)‐adenosine 5′‐triphosphate (BzATP)‐induced release of previously incorporated [3H]GABA. Both Brilliant Blue G and oxidized ATP inhibited the release of [3H]GABA caused by ATP application; the Brilliant Blue G‐sensitive, P2X7 receptor‐mediated fraction, was much larger after ischemia than after normoxia. Whereas ischemic stimulation failed to alter the amplitude of ATP‐ and BzATP‐induced small inward currents recorded from a subset of non‐pyramidal neurons, BzATP caused a more pronounced increase in the frequency of miniature inhibitory postsynaptic currents (mIPSCs) after ischemia than after normoxia. Brilliant Blue G almost abolished the effect of BzATP in normoxic neurons. Since neither the amplitude of mIPSCs nor that of the muscimol‐induced inward currents was affected by BzATP, it is assumed that BzATP acts at presynaptic P2X7 receptors. Finally, P2X7 receptors did not enhance the intracellular free Ca2+ concentration either in proximal dendrites or in astrocytes, irrespective of the normoxic or ischemic pre‐incubation conditions. Hence, facilitatory P2X7 receptors may be situated at the axon terminals of GABAergic non‐pyramidal neurons. When compared with normoxia, ischemia appears to markedly increase P2X7 receptor‐mediated GABA release, which may limit the severity of the ischemic damage. At the same time we did not find an accompanying enhancement of P2X7 mRNA or protein expression, suggesting that receptors may become hypersensitive because of an increased efficiency of their transduction pathways.

Neuroprotection by ATP-dependent potassium channels in rat neocortical brain slices during hypoxia.

Morphological changes induced by 30 min of hypoxia (incubation in medium saturated with 95% N2-5% CO2 instead of the normal 95% O2-5% CO2) were investigated in neurons (layers II/III of the parietal cortex) of rat neocortical brain slices. The cells were identified as intact, reversibly or irreversibly injured. As expected, hypoxia decreased the number of intact cells and increased the number of irreversibly injured cells. Pretreatment of slices with diazoxide (300 microM), an agonist of ATP-dependent potassium (KATP) channels completely prevented the morphological damage induced by hypoxia, whereas tolbutamide (300 microM), an antagonist of KATP channels, was ineffective when given alone. However, tolbutamide (300 microM) co-applied with diazoxide (300 microM), partly reversed the neuroprotective effect of this agonist during hypoxia. In conclusion, KATP channels appear to be present on neocortical neurons and their opening counteracts hypoxia-induced cell injury.