Ectonucleotidases in Müller glial cells of the rodent retina: Involvement in inhibition of osmotic cell swelling (original) (raw)
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Immunocytochemical localization of NTPDases1 and 2 in the neural retina of mouse and zebrafish
Synapse, 2009
Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) are a family of membrane-bound enzymes that hydrolyze extracellular di-and triphosphate nucleosides. E-NTPDases have been proposed to control extracellular nucleotide levels that mediate intercellular communication by binding to specific membrane receptors. Here we show a detailed immunocytochemical localization of two enzymes of the E-NTPDase family in the retinal layers of two vertebrate species, namely, the mouse and the zebrafish. In the mouse retina, NTPDase2 was chiefly localized in Mü ller glia and ganglion cell processes. NTPDase1 was located on neurons as well, since it was expressed by horizontal and ganglion cell processes, suggesting that nucleotides such as ATP and ADP can be hydrolyzed at the surface of these cells. NTPDase1 was also detected in intraretinal blood vessels of the mouse. Regarding zebrafish, NTPDases1 and 2 seem to be differentially localized in horizontal cell processes, photoreceptor segments, and ganglion cell dendrites and axons, but absent from Mü ller glia. Moreover, NTPDases1 and 2 appear to be expressed within the germinal margin of the zebrafish retina that contains proliferative and differentiating cells. Retinal homogenates from both species exhibited ecto-ATPase activity which might be attributed at least to NTPDases1 and 2, whose expression is described in this report. Our results suggest a compartmentalized regulation of extracellular nucleotide/nucleoside concentration in the retinal layers, supporting a relevant role for extracellular nucleotide mediated-signaling in vertebrate retinas.
Endogenous purinergic signaling is required for osmotic volume regulation of retinal glial cells
Journal of Neurochemistry, 2010
Neuronal activity is associated with rapid ion shifts between intra-and extracellular spaces (ECS) and changes in the cellular and ECS volumes. In the sensory retina, activation of neuronal ionotropic glutamate receptors has been shown to cause a net uptake of sodium chloride, resulting in a swelling of neuronal cell bodies and synapses, and a decrease in the ECS volume . During intense neuronal activity, retinal cells are surrounded by a hypoosmotic environment. Light-evoked changes in the ionic composition of the ECS fluid cause a decrease in osmolarity of the ECS fluid, because the decrease in sodium exceeds the increase in potassium by a factor of two, which is accompanied by a decrease in the chloride concentration ). An activity-dependent decrease in osmolarity of the ECS fluid was also observed in the brain . Retinal glial cells take up neuronderived osmolytes such as potassium and sodium-glutamate , which may elevate the intracellular space; IP 3 R2, inositol 1,4,5-triphosphate receptor type 2; MRS2179, N 6 -methyl-2¢-deoxyadenosine-3¢,5¢-bisphosphate; MRS2365, (N)-methanocarba-2MeSADP.
Ophthalmic Research, 2003
During proliferative vitreoretinopathy (PVR) Müller glial cells show an up-regulation of their responsiveness to extracellular adenosine 5)-triphosphate (ATP). In the present study, we investigated if such a glial cell response is also a feature for other retinopathies besides PVR. To this aim, the proteolytic enzyme, dispase (0.1 U), was injected into the vitreous of rabbit eyes. After 3 weeks, a distinct retinopathy had developed which showed no signs of PVR. The retinopathy was characterized by strong alterations of the retinal vasculature in the medullary rays, by photoreceptor degeneration, retinal atrophy, and activation of microglial cells. Müller cells became reactive, as indicated by up-regulation of glial fibrillary acidic protein immunoreactivity and by hypertrophy involving subretinal fibrosis. Müller cell reactivity was also evidenced electrophysiologically by a downregulation of their inwardly rectifying potassium currents and by an up-regulation of their responsiveness to extracellular ATP. Significantly more Müller cells from dispase-treated eyes showed ATP-evoked calcium (83%) and current responses (69%) when compared with cells from control eyes (13 and 9%, respectively). The results indicate that increased responsiveness to extracellular ATP may be a more general feature of Müller cell gliosis, and is also observed in retinopathies besides PVR.
Neurochemical research, 2015
Retinal glial (Müller) cells release ATP upon osmotic stress or activation of metabotropic glutamate receptors. ATP inhibits the osmotic Müller cell swelling by activation of P2Y1 receptors. In the present study, we determined the molecular pathways of the ATP release from Müller cells in slices of the rat retina. Administration of the ATP/ADPase apyrase induced a swelling of Müller cells under hypoosmotic conditions, and prevented the swelling-inhibitory effect of glutamate, suggesting that swelling inhibition is mediated by extracellular ATP. A hypoosmotic swelling of Müller cells was also observed in the presence of a blocker of multidrug resistance channels (MK-571), a CFTR inhibitor (glibenclamide), and connexin hemichannel blockers (18-α-glycyrrhetinic acid, 100 µM carbenoxolone). The swelling-inhibitory effect of glutamate was prevented by MK-571, the connexin hemichannel blockers, and a pannexin-1 hemichannel blocker (5 µM carbenoxolone). The p-glycoprotein blocker verapamil...
Involvement of A(1) adenosine receptors in osmotic volume regulation of retinal glial cells in mice
Molecular vision, 2009
Osmotic swelling of Müller glial cells has been suggested to contribute to retinal edema. We determined the role of adenosine signaling in the inhibition of Müller cell swelling in the murine retina. The size of Müller cell somata was recorded before and during perfusion of retinal sections and isolated Müller cells with a hypoosmolar solution. Retinal tissues were freshly isolated from wild-type mice and mice deficient in A(1) adenosine receptors (A(1)AR(-/-)), or cultured as whole-mounts for three days. The potassium conductance of Müller cells was recorded in isolated cells, and retinal slices were immunostained against Kir4.1. Hypotonic exposure for 4 min induced a swelling of Müller cell bodies in retinal slices from A(1)AR(-/-) mice but not wild-type mice. Pharmacological inhibition of A(1) receptors or of the ecto-5'-nucleotidase induced hypoosmotic swelling of Müller cells from wild-type mice. Exogenous adenosine prevented the swelling of Müller cells from wild-type but ...
1997
The presence of receptors for ATP has not been established in any native preparation of retinal neurons or glia. In the present study, we used conventional electrophysiological and [Ca 2ϩ ] in fluorescence imaging techniques to investigate the effects of ATP added to Ringer's solution perfusing the retinal-facing (apical) membrane of freshly isolated monolayers of bovine retinal pigment epithelium (RPE). ATP (or UTP) produced large, biphasic voltage and resistance changes with a K d of ϳ5 M for ATP and ϳ1 M for UTP. Electrical and pharmacological evidence indicates that the first and second phases of the response are attributable to an increase in basolateral membrane Cl conductance and a decrease in apical membrane K conductance, respectively. The ATP-induced responses were not affected by adenosine, but were reduced by the P 2 -purinoceptor blocker suramin. ATP also produced a large, transient increase in [Ca 2ϩ ] in that was blocked by cyclopiazonic acid, an inhibitor of endoplasmic reticulum Ca 2ϩ -ATPases. The calcium buffer BAPTA attenuated the voltage effects of ATP. We also found that apical DIDS significantly inhibited the ATP-evoked [Ca 2ϩ ] in and electrical responses, suggesting that DIDS blocked the purinoceptor. Measurements of fluid movement across the RPE using the capacitance probe technique demonstrated a significant increase in fluid absorption by apical UTP. These data indicate the presence of metabotropic P 2Y /P 2U -purinoceptors at the RPE apical membrane and implicate extracellular ATP in vivo as a retinal signaling molecule that could help regulate the hydration and chemical composition of the subretinal space.
Investigative ophthalmology & visual science, 2002
To test whether in an animal model of proliferative vitreoretinopathy (PVR) the Müller glial cells displayed an upregulation of purinergic P2 receptor-mediated responses. PVR was induced by intravitreal injection of the proteolytic enzyme, dispase, in the eyes of adult rabbits. The developing PVR was examined ophthalmoscopically. After 3 weeks, small retinal pieces were wholemounted and used for calcium imaging, freshly dissociated Müller cells were subjected to calcium imaging, and patch-clamp recordings were made. The presence of P2 receptor-mediated Ca(2+) responses was determined both directly--that is, fluorometrically--and indirectly, by electrophysiological recording of Ca(2+)-activated K(+) currents. According to earlier observations in another model of retinal detachment and PVR, the reactive Müller cells displayed hypertrophy, downregulation of inwardly rectifying K(+) currents, and depolarization of the resting membrane potential, all dependent on the severity of the PVR....
Journal of Molecular Medicine, 2019
ATP and adenosine are important signaling molecules involved in vascular remodeling, retinal function, and neurovascular coupling in the eye. Current knowledge on enzymatic pathways governing the duration and magnitude of ocular purinergic signaling is incompletely understood. By employing sensitive analytical assays, this study dissected ocular purine homeostasis as a complex and coordinated network. Along with previously characterized ecto-5′-nucleotidase/CD73 and adenylate kinase activities, other enzymes have been identified in vitreous fluids, including nucleoside triphosphate diphosphohydrolase (NTPDase), adenosine deaminase, and alkaline phosphatase. Strikingly, activities of soluble adenylate kinase, adenosine deaminase, ecto-5′-nucleotidase/CD73, and alkaline phosphatase, as well as intravitreal concentrations of ATP and ADP, were concurrently upregulated in patients suffering from diabetic retinopathy (DR) with non-clearing vitreous hemorrhage (VH), when compared to DR eyes without VH and control eyes operated due to macular hole or pucker. Additional histochemical analysis revealed selective distribution of key ecto-nucleotidases (NTPDase1/CD39, NTPDase2, ecto-5′-nucleotidase/CD73, and alkaline phosphatase) in the human sensory neuroretina and optic nerve head, and also in pathological neofibrovascular tissues surgically excised from patients with advanced proliferative DR. Collectively, these data provide evidence for specific hemorrhage-related shifts in purine homeostasis in DR eyes from the generation of anti-inflammatory adenosine towards a pro-inflammatory and proangiogenic ATP-regenerating phenotype. In the future, identifying the exact mechanisms by which a broad spectrum of soluble and membrane-bound enzymes coordinately regulates ocular purine levels and the further translation of purine-converting enzymes as potential therapeutic targets in the treatment of proliferative DR and other vitreoretinal diseases will be an area of intense interest.
Regulation of Extracellular Atp in the Zebrafish Retina: Ectonucleotidases Localization and Activity
2005
ATP is released by neurons and acts as a neurotransmitter andneuromodulator via purinergic receptors. Extracellular ATPconcentrations are precisely regulated by ecto-nucleosidetriphosphatediphosphohydrolases (ENTPDases). Resulting AMPis further metabolized extracellularly to adenosine, a potentinhibitory neuromodulator. We showed that two major subtypes ofectonucleotidases: ENTPDase 1 and 2 are expressed in the zebrafishretina both by westem blot and imrriunohistochemistry on 10 umtissue sections. Both enzymes are heterogeneously distributedamong retinallayers, principally expressed in the inner half oftheretina but their expression pattern overlaps only partially.ENTPDases 1 and 2 activities can be distinguished one from eachother by autoradiography using radioactive nucleotides as substrates.To date, we demonstrated extracellular ATPase activity on zebrafishretinal sections by this methodology. We are currently comparingwhether the site of ATP hydrolysis match es the immunoreactiv...