Biomedical subjects

Andreas Bringmann

Publications and source records attributed to Andreas Bringmann.

At least 55 records · Page 3Linked to original sources

PubMed2004

GABA(A) receptors in Müller glial cells of the human retina.

The present study was aimed at characterizing the GABA(A) receptor-mediated currents in acutely isolated glial (Müller) cells of the human retina and investigating their subcellular localization across the Müller cell membrane. Extracellular application of GABA evoked two current responses in human Müller cells: a fast transient GABA(A) receptor-mediated current that inactivated within 10 s and that was independent of extracellular Na(+), and a sustained current that was dependent on extracellular Na(+) and that was mediated by high-affinity GABA transporters. The receptor current was half-maximally activated at a GABA concentration of 32 microM, while the transporter current showed an affinity constant of 7.9 microM GABA. The receptor currents were blocked by bicuculline and picrotoxin and were also activated by muscimol or by other amino acids. The receptor currents are Cl(-) currents, as indicated by the close relationship between the reversal potential of these currents and the Cl(-) equilibrium potential. Using perforated-patch recordings, a mean intracellular Cl(-) concentration of 37 +/- 12 mM was determined in human Müller cells. Using electrophysiological and fluorescence imaging methods, it was revealed that GABA(A) receptors are unevenly distributed across the Müller cell membrane, with higher densities at the endfoot, at the soma, and at the distal sclerad end of the cells. It is concluded that GABA(A) receptor expression may allow a sensing of retinal GABAergic neuronal signal transmission by Müller cells.

Action Potentials↗

PubMed2004

A potassium channel-linked mechanism of glial cell swelling in the postischemic retina.

The cellular mechanisms underlying glial cell swelling, a central cause of edema formation in the brain and retina, are not yet known. Here, we show that glial cells in the postischemic rat retina, but not in control retina, swell upon hypotonic stress. Swelling of control cells could be evoked when their K(+) channels were blocked. After transient ischemia, glial cells strongly downregulated their K(+) conductance and their prominent Kir4.1 protein expression at blood vessels and the vitreous body. In contrast, the expression of the aquaporin-4 (AQP4) (water channel) protein was only slightly altered after ischemia. Activation of D(2) dopaminergic receptors prevents the hypotonic glial cell swelling. The present results elucidate the coupling of transmembraneous water fluxes to K(+) currents in glial cells and reveal the role of altered K(+) channel expression in the development of cytotoxic edema. We propose a mechanism of postischemic glial cell swelling where a downregulation of their K(+) conductance prevents the emission of intracellularly accumulated K(+) ions, resulting in osmotically driven water fluxes from the blood into the glial cells via aquaporins. Inhibition of these water fluxes may be beneficial to prevent ischemia-evoked glial cell swelling.

Animals↗

PubMed2004

Glial cell expression of hepatocyte growth factor in vitreoretinal proliferative disease.

The hepatocyte growth factor (HGF) has been crucially implicated in the development of proliferative retinal diseases; however, it is unclear whether retinal glial cells express or respond to HGF. Therefore, we examined the expression of HGF and of the receptor for HGF, c-Met, by immunohistochemical costaining with glial fibrillary acidic protein (GFAP) in epiretinal membranes of patients with proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), respectively. Furthermore, it was determined whether cells of the human retinal glial cell line, MIO-M1, secrete HGF protein, and whether HGF stimulates proliferation and chemotaxis, and secretion of the vascular endothelial growth factor (VEGF). Neuroretinas of patients with PVR express elevated mRNA level for HGF in comparison to control retinas. In epiretinal membranes of patients with PVR or PDR, immunoreactivity for HGF and for c-Met, respectively, partially colocalized with immunoreactivity for GFAP. Fetal bovine serum and basic fibroblast growth factor, but not heparin-binding epidermal or platelet-derived growth factors, evoked HGF secretion by cultured retinal glial cells. HGF displayed only a marginal effect on cell proliferation while it stimulated chemotaxis. HGF promoted the secretion of VEGF, via activation of the phosphatidylinositol-3 kinase. It is concluded that glial cells in epiretinal membranes express both HGF protein and c-Met receptors. The results suggest an autocrine/paracrine role of HGF in glial cell responses during proliferative vitreoretinal disorders as well as in retinal neovascularization, by stimulating of VEGF release.

Adolescent↗

PubMed2003

Upregulation of purinergic P2Y receptor-mediated calcium responses in glial cells during experimental detachment of the rabbit retina.

To investigate injury-induced alterations of purinergic P2Y receptor-mediated calcium responses in glial (Müller) cells of the rabbit retina, neural retinae were experimentally detached from the pigment epithelium. The ATP-evoked calcium responses were recorded in the endfeet of glial cells at the vitread surface of retinal wholemounts. In control retinae, approximately 7% of the glial cells investigated showed ATP-evoked calcium responses. Within 24 h of detachment, significantly more retinal glial cells (42%) showed calcium responses, and glial ATP responsiveness increased further in retinae which were detached for 48 (44%) or for 72 h (64%). The results indicate that in the detached retina, glial cells upregulate their responsiveness to extracellular ATP within 24 h of injury. Thus, P2Y receptor-mediated signalling may be involved in the early steps of glial response to retinal injury.

Animals↗

PubMed2003

Age-related decrease of potassium currents in glial (Müller) cells of the human retina.

BACKGROUND: Age-dependent alterations have been investigated far less in retinal glial cells than in retinal neurons. We investigated age-dependent alterations of inwardly rectifying potassium (Kir) currents in Müller glial cells of the human retina. METHODS: Müller cells were isolated immediately post mortem from donors without a reported history of eye disease, and the amplitudes of Kir currents and of currents through high-voltage-activated (HVA) calcium channels were measured by whole-cell patch clamping. RESULTS: The amplitude of the Kir currents was lower in the cells from donors older than 50 years than in the cells of younger donors; the decrease was strongly correlated with the donor's age (p < 0.001). The current amplitude in the cells from donors older than 60 years was about 40% lower than the amplitude in the cells from donors younger than 50 years. The amplitude of the HVA currents was greater in the cells from donors older than 55 years than in the cells from younger donors; the increase, up to about 500%, was strongly age-dependent (p < 0.001). INTERPRETATION: The age-related decrease in Kir-current amplitude in Müller cells may reflect the neuron loss in the aged retina. Our findings also indicate that retinal glial cells have enhanced cytoplasmic calcium signals in the course of aging.

Adult↗

PubMed2003

Diffuse perineuronal nets and modified pyramidal cells immunoreactive for glutamate and the GABA(A) receptor alpha1 subunit form a unique entity in rat cerebral cortex.

Perineuronal nets (PNs) consisting of polyanionic chondroitin sulfate proteoglycans (CSPG) and other extracellular matrix components create an exceptional microenvironment around certain types of neurons. In rat neocortex, three types of PNs can be distinguished after staining with Wisteria floribunda agglutinin (WFA) by their different morphological structure: lattice-like PNs associated with subpopulations of nonpyramidal neurons, weakly labeled PNs showing a pyramidal morphology, and diffuse PNs that possess a thick, strongly labeled matrix sheath located mainly in layer VIb above the white matter. The type of neuron surrounded by diffuse nets has not been described so far. This study is focused on the cytochemical and morphological characteristics of neurons associated with diffusely contoured PNs in rat parietal cortex using immunocytochemical staining, intracellular injection, and retrograde tracing methods. Cells surrounded by diffuse PNs were glutamate-immunoreactive in contrast to nonpyramidal, net-associated neurons that showed immunoreactivity for GABA, the calcium-binding protein parvalbumin and the potassium channel subunit Kv3.1b. Both groups of PN-ensheathed cells were mostly immunoreactive for the GABA(A) receptor alpha1 subunit. Lucifer Yellow-injected neurons surrounded by diffuse PNs displayed the morphological properties of modified pyramidal cells with intracortical main axons. Many neurons with diffuse PNs were retrogradely labeled over a long distance after Fluoro-Gold tracer injection in the parietal cortex, but remained unlabeled after intrathalamic injection. We conclude that neurons associated with diffuse PNs are a subpopulation of glutamatergic modified pyramidal cells that could act as excitatory long-range intracortically projecting neurons.

Animals↗

PubMed2003

P2Y receptor-mediated stimulation of Müller glial cell DNA synthesis: dependence on EGF and PDGF receptor transactivation.

PURPOSE: To determine whether P2Y receptor-evoked proliferation of Müller glial cells depends on transactivation of receptor tyrosine kinases. METHODS: Primary cultures of Müller cells of the guinea pig were treated with test substances for 16 hours. The DNA synthesis rate was assessed by a bromodeoxyuridine (BrdU) immunoassay, and the phosphorylation states of the extracellular signal-regulated kinase (ERK1/2) and the p38 mitogen-activated protein kinase (p38 MAPK) were determined by Western blot analysis. RESULTS: In Müller cells, the mitogenic effect of P2Y receptor activation by extracellular adenosine triphosphate (ATP) depended on transactivation of both the platelet-derived growth factor (PDGF) and the epidermal growth factor (EGF) receptor tyrosine kinases, as suggested by the blocking effects of the tyrphostins AG1296 and AG1478 on the ATP-induced proliferation and phosphorylation of ERK1/2. Moreover, the PDGF-induced proliferation may depend on transactivation of the EGF receptor kinase. Antibodies against heparin-binding EGF (HB-EGF) or PDGF, as well as inhibition of matrix metalloproteinases (MMPs) blocked ATP-evoked proliferation. At least one metalloproteinase (MMP-9), was implicated in the signal transfer from P2Y to EGF receptors. In contrast, the mitogenic effect of fetal calf serum was independent of growth factor receptor activity. P2Y receptor activation stimulated Müller cell proliferation by activating the ERK1/2 and the phosphatidylinositol 3 (PI3) kinase signaling pathways, whereas the p38 MAPK pathway was not involved in mitogenic signaling. CONCLUSIONS: The present data suggest that P2Y-receptor-induced mitogenic signaling in Müller cells is mediated by transactivation of the PDGF and EGF receptor tyrosine kinases. The transactivation may be mediated by release of PDGF and MMP-dependent shedding of HB-EGF from the Müller cell matrix, respectively. The transactivation of the receptor tyrosine kinases may result in activation of ERK1/2 and PI3 kinase and an increase in the proliferation rate.

Adenosine Triphosphate↗

PubMed2003

Early glial cell reactivity in experimental retinal detachment: effect of suramin.

PURPOSE: In a rabbit model of retinal detachment, early Müller glial cell reactivity was monitored-specifically, changes in membrane features-to determine whether these changes involve an upregulation of purinergic P2 receptor-mediated responses and whether all or some of these alterations could be blocked by suramin or pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS). In addition, the immune cell reactivity (microglial cells and blood-derived immune cells) was monitored. METHODS: A local retinal detachment was induced by subretinal injection of a sodium hyaluronate solution. Three, 24, 48, and 72 hours after surgery, Müller cells were acutely isolated, and patch-clamp records of the whole-cell potassium currents were made. The presence of P2 receptor-mediated responses was determined by measuring extracellular adenosine triphosphate (ATP)-induced membrane current increases, and by recording of ATP-induced calcium responses at the vitreal surface of retinal wholemounts. The density of isolectin B(4)-labeled immune cells was determined in the nerve fiber layer of retinal wholemounts. RESULTS: Within 24 hours of detachment, Müller cell reactivity was evident. The cells downregulated the density of their inwardly rectifying potassium currents to 60% and 47% of the control value at 48 hours and 72 hours of detachment, respectively. This downregulation was accompanied by an enhanced incidence of cells which showed calcium and current responses after ATP application (control: 14%; 24 hours of detachment: 42%; 72 hours of detachment: 80%). Müller cell hypertrophy was apparent at 48 and 72 hours of detachment. Application of suramin during surgery inhibited the downregulation of potassium currents, but not the elevated responsiveness to extracellular ATP; PPADS had no effect. Suramin also inhibited the inflammatory response that was induced by the surgical procedure and that was apparent by the increased number of immune cells. CONCLUSIONS: Reactive responses of Müller cells occur within 24 hours of detachment. Suramin inhibits several (but not all) reactive glial alterations and therefore may represent one candidate for further investigations in the search for drugs that limit detrimental effects of immune cell activation and Müller cell gliosis during retinal detachment.

Adenosine Triphosphate↗

PubMed2002

ATP-evoked calcium responses of radial glial (Müller) cells in the postnatal rabbit retina.

Here we show that rabbit Müller cell differentiation from radial glial progenitor cells is accompanied by a decreasing capability to respond to specific stimuli (depolarization and extracellular adenosine 5'-triphosphate [ATP]) with an elevation of intracellular calcium. Intracellular free calcium was recorded in retinal wholemounts from young (postnatal days [P] 2 to 31) and adult rabbits. Images were taken from the nerve fiber/ganglion cell layers where the endfeet of radial glial/ Müller cells can be identified after selective uptake of calcium-sensitive dyes. The area of responding endfeet was determined as the percentage of the total area occupied by Müller cell endfeet, as an estimate of the percentage of responding cells. In response to depolarization (50 mM potassium), an increase of intracellular free calcium occurred in 19% of cells from young postnatal retinae (P2-31) but only in 2% from adults. This depolarization-induced calcium rise was caused both by a calcium influx from extracellular space and by an intracellular calcium release. The latter response was inhibited by the P2 receptor blocker pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid (PPADS), indicating that extracellular calcium-independent ATP release into the extracellular space occurs during retinal depolarization. When extracellular ATP (200 microM) was applied, calcium responses were recorded in 83% of cells from young postnatal retinae (P2-6); in the course of further development, both the percentage of responding cells (7% in retinae from adult rabbits) and the amplitude of the calcium responses decreased. It is concluded that during the differentiation of immature radial glia into mature Müller cells, stimulus-evoked intracellular calcium signaling mechanisms change.

Adenosine↗

PubMed2002

Diversity of Kir channel subunit mRNA expressed by retinal glial cells of the guinea-pig.

One of the main functions of Müller glial cells is the performance of retinal K+ homeostasis which is thought to be primarily mediated by K+ fluxes through inwardly rectifying K+ (Kir) channels expressed in Müller cell membranes. Until now, there is limited knowledge about the types of Kir channel subunits expressed by Müller cells. Using RT-PCR, we investigated the expression of mRNA encoding different Kir channel subunits in the retina of the guinea pig. In order to verify expression by Müller cells, primary cultures of guinea pig Müller cells were also investigated. Both retinae and cultured Müller cells express mRNA for a diversity of Kir channel subtypes which include members of at least four channel subfamilies: Kir2.1, Kir2.2, Kir2.4, Kir3.1, Kir 3.2, Kir4.1, Kir6.1, and Kir6.2. mRNAs for the following Kir channel subtypes were not detected in Müller cells: Kir1.1, Kir2.3, Kir3.3, Kir3.4, Kir4.2, and Kir5.1. It is concluded that the spatial buffering of extracellular K+ by Müller cells may be mediated by cooperation of different subtypes of Kir channels, and that the distinct Kir channel types involved in this function may change depending on the physiological or metabolic state of the retina.

Animals↗

PubMed2002

Activation of P2Y receptors stimulates potassium and cation currents in acutely isolated human Müller (glial) cells.

The ability of various neurotransmitters/neuroactive substances to induce fast, transient rises of Ca(2+)-activated K(+) currents (I(BK)) caused by release of Ca(2+) from intracellular stores was investigated in Müller glial cells of the human retina. Müller cells were enzymatically isolated from retinas of healthy donors or of patients with proliferative vitreoretinopathy, and the transmembrane ionic currents were recorded using the whole-cell and the cell-attached patch-clamp techniques. The results of the screening experiments indicate that human Müller cells express, in addition to GABA(A) and perhaps glutamatergic and cholinergic receptors, predominantly P2 receptors. ATP and other nucleotides exerted two effects on membrane currents: repetitive transient increases of the I(BK) amplitude and, in a subpopulation of cells investigated, the appearance of a transient cation conductance at negative potentials. ATP and UTP increased dose-dependently the I(BK) amplitude with half-maximal effects at 0.33 and 0.50 microM, respectively. Since several different P2 receptor agonists increased the I(BK), it is assumed that human Müller cells express a mixture of different types of P2Y receptors. In cell-attached patches, extracellular application of ATP or UTP transiently increased the open probability of single putative BK channels. The increase of I(BK) and the appearance of the cation conductance in whole-cell records were abolished when intracellular Ca(2+) was buffered by a high-EGTA pipette solution or when IP(3) was included in the pipette solution. The expression of agonist-evoked transient cation currents was found to be stronger in cells from patients as compared to cells from healthy donors. It is concluded that human Müller glial cells express P2Y receptors that, via IP(3) formation, cause intracellular Ca(2+) release. The increased intracellular Ca(2+) concentration stimulates the activity of BK channels and may induce opening of cation channels. Both the ATP-induced activity of BK channels and the increased expression of Ca(2+)-gated cation channels may be important in respect to proliferative Müller cell gliosis.

Adenosine Triphosphate↗

PubMed2002

Electrophysiological characterization of retinal Müller glial cells from mouse during postnatal development: comparison with rabbit cells.

The electrophysiology of murine Müller cells, and of their precursors, during postnatal development was investigated by using the whole-cell patch-clamp technique. Membrane potential, membrane capacitance, and expression of voltage-gated Na+ currents increased during the first 3 postnatal weeks. During the same period, the membrane resistance decreased due to an upregulation of both inward and outward K+ currents. Glutamate transporter-mediated currents increased during the first postnatal weeks, as well. In Müller (precursor) cells from rabbits, these transporter currents already achieved adult levels at postnatal day 6, the earliest stage studied. Together with the developmental time course of K+ currents, this indicates a delay in the maturation of murine Müller cells, as compared with cells from rabbit.

Aging↗

PubMed2002

Functional expression of Kir 6.1/SUR1-K(ATP) channels in frog retinal Müller glial cells.

The retinae and brains of larval and adult amphibians survive long-lasting anoxia; this finding suggests the presence of functional K(ATP) channels. We have previously shown with immunocytochemistry studies that retinal glial (Müller) cells in adult frogs express the K(ATP) channel and receptor proteins, Kir6.1 and SUR1, while retinal neurons display Kir6.2 and SUR2A/B (Skatchkov et al., 2001a: NeuroReport 12:1437-1441; Eaton et al., in press: NeuroReport). Using both immunocytochemistry and electrophysiology, we demonstrate the expression of Kir6.1/SUR1 (K(ATP)) channels in adult frog and tadpole Müller cells. Using conditions favoring the activation of K(ATP) channels (i.e., ATP- and spermine-free cytoplasm-dialyzing solution containing gluconate) in Müller cells isolated from both adult frogs and tadpoles, we demonstrate the following. First, using the patch-clamp technique in whole-cell recordings, tolbutamide, a blocker of K(ATP) channels, blocks nearly 100% of the transient and about 30% of the steady-state inward currents and depolarizes the cell membrane by 5-12 mV. Second, inside-out membrane patches display a single-channel inward current induced by gluconate (40 mM) and blocked by ATP (200 microM) at the cytoplasmic side. The channels apparently show two sublevels (each of approximately 27-32 pS) with a total of 85-pS maximal conductance at -80 mV; the open probability follows a two-exponential mechanism. Thus, functional K(ATP) channels, composed of Kir6.1/SUR1, are present in frog Müller cells and contribute a significant part to the whole-cell K+ inward currents in the absence of ATP. Other inwardly rectifying channels, such as Kir4.1 or Kir2.1, may mediate the remaining currents. K(ATP) channels may help maintain glial cell functions during ATP deficiency.

ATP-Binding Cassette Transporters↗

PubMed2002

High-affinity GABA uptake in retinal glial (Müller) cells of the guinea pig: electrophysiological characterization, immunohistochemical localization, and modeling of efficiency.

Glial cells may act as important modulators of neuronal information processing, in particular, via fast uptake of neuronally released transmitters. Here, we characterize the electrogenic gamma-aminobutyric acid (GABA) transporters present in the plasma membranes of Müller (glial) cells of the guinea pig retina and present an estimate of their functional efficiency. The GABA-evoked whole-cell currents are voltage-dependent, with increasing amplitudes and decreasing affinity constants at more negative membrane potentials. The transmembranal GABA transport is concentration-dependent, with near-maximal currents at 100 microM GABA, and is dependent on extracellular sodium and chloride ions; the stoichiometry is 1 GABA/2 Na(+)/1 Cl(-). Immunohistochemical labeling and whole-cell voltage-clamp records reveal that Müller cells express both GAT-1 and GAT-3 (but not GAT-2), and that the transporter proteins are expressed predominantly at plasma membrane sites that, in situ, are localized in the outer retina where GABA uptake is performed exclusively by Müller cells. When extracellular GABA enters the cell interior, it evokes, via activation of the GABA transaminase, an NAD(P)H fluorescence signal selectively in the distal region of the Müller cells where their mitochondria are located. Using our experimental data, we simulated the GABA clearance from the extracellular space surrounding one Müller cell; these estimates show that a pulse of 100 microM extracellular GABA is fully cleared after 70 ms. It is suggested that Müller cells may be involved in the regulation of GABAergic transmission within the retina by providing a fast termination of GABAergic signaling via their highly efficient GABA uptake.

Action Potentials↗

PubMed2002

Kir potassium channel subunit expression in retinal glial cells: implications for spatial potassium buffering.

To understand the role of different K(+) channel subtypes in glial cell-mediated spatial buffering of extracellular K(+), immunohistochemical localization of inwardly rectifying K(+) channel subunits (Kir2.1, Kir2.2, Kir2.3, Kir4.1, and Kir5.1) was performed in the retina of the mouse. Stainings were found for the weakly inward-rectifying K(+) channel subunit Kir4.1 and for the strongly inward-rectifying K(+) channel subunit Kir2.1. The most prominent labeling of the Kir4.1 protein was found in the endfoot membranes of Müller glial cells facing the vitreous body and surrounding retinal blood vessels. Discrete punctate label was observed throughout all retinal layers and at the outer limiting membrane. By contrast, Kir2.1 immunoreactivity was located predominantly in the membrane domains of Müller cells that contact retinal neurons, i.e., along the two stem processes, over the soma, and in the side branches extending into the synaptic layers. The results suggest a model in which the glial cell-mediated transport of extracellular K(+) away from excited neurons is mediated by the cooperation of different Kir channel subtypes. Weakly rectifying Kir channels (Kir4.1) are expressed predominantly in membrane domains where K(+) currents leave the glial cells and enter extracellular "sinks," whereas K(+) influxes from neuronal "sources" into glial cells are mediated mainly by strongly rectifying Kir channels (Kir 2.1). The expression of strongly rectifying Kir channels along the "cables" for spatial buffering currents may prevent an unwarranted outward leak of K(+), and, thus, avoid disturbances of neuronal information processing.

Animals↗

PubMed2002

Membrane conductance of Müller glial cells in proliferative diabetic retinopathy.

BACKGROUND: It is not known whether the membrane features of human Müller cells are altered in proliferative diabetic retinopathy (PDR). We performed a study to investigate the expression of several distinct forms of membrane conductance in Müller glial cells from a patient with PDR compared to cells from healthy donors (control cells). METHODS: Müller cells were isolated 2 hours after vitreoretinal surgery in the case of the patient and within 24 hours in the case of the autopsy eyes. Whole-cell voltage-clamp recordings were made. The results for the two groups were compared with the Mann-Whitney U test. RESULTS: As assayed by the whole-cell membrane capacitance, the cells from the patient with PDR showed hypertrophy in comparison to the control cells (mean 85.1 pF [standard deviation (SD) 19.7 pF] vs. 54.3 pF [SD 13.8 pF]). The cells from the patient displayed strong downregulation of inwardly rectifying potassium ion (Kir) currents (mean 0.41 [SD 0.24] pA/pF, compared to 3.43 [SD 1.86] pA/pF for the control cells). The Kir current downregulation was accompanied by a less negative membrane potential (-57.3 mV [SD 16.9 mV], compared with -82.3 mV [SD 5.3 mV] for the control cells). Both the number and the amplitude of voltage-gated sodium ion currents were enhanced in cells from the patient. When P2X7 receptors were activated by 2'-/3'-O-(4-benzoylbenzoyl)-adenosine triphosphate, cells in both groups displayed opening of a cation conductance and, simultaneously, an increase in currents through calcium ion-activated potassium ion channels. INTERPRETATION: Changes in Müller cell membrane conductance in PDR are similar to those described in proliferative vitreoretinopathy. The down-regulation of active Kir channels and the membrane depolarization likely disturb voltage-dependent Müller cell functions, such as regulation of local ion concentrations and uptake of neurotransmitters. The enhanced entry of calcium ions from the extracellular space and the subsequent stimulation of calcium-activated potassium channels support Müller cell proliferation in PDR.

Adenosine Triphosphate↗

PubMed2002

P2Y receptor-mediated stimulation of Müller glial DNA synthesis.

PURPOSE: To determine whether activation of P2Y receptors may increase the DNA synthesis rate of cultured Müller cells and to investigate whether adenosine 5'-triphosphate (ATP)-induced Müller cell proliferation is mediated by an intracellular calcium increase. METHODS: Primary cultures of Müller cells of the guinea pig were treated with test substances for 16 hours. The DNA synthesis rate was assessed by a bromodeoxyuridine immunoassay, and ATP-induced elevations of the intracellular calcium concentration were recorded by fura-2 imaging. RESULTS: ATP or uridine triphosphate (UTP) increased the DNA synthesis rate whereas alpha,beta-methylene-ATP, 2-methyl-thio-ATP, and adenosine were ineffective, indicating that the action of ATP was through P2Y receptors. The effect of ATP was dose dependent, with an EC(50) of 5.9 microM. The mitogenic effect of ATP required an elevation of the intracellular calcium and a calcium influx into Müller cells. Blockers of calcium-permeable channels (nickel ions) or of calcium-dependent potassium (BK) channels (iberiotoxin, charybdotoxin) inhibited the ATP-stimulated DNA synthesis. In calcium-imaging experiments, ATP-evoked intracellular calcium transients were significantly shortened in the presence of extracellular nickel ions or of iberiotoxin. A correlation was found between the duration of the ATP-evoked calcium transients and the basal proliferation rate of the cultures. CONCLUSIONS: The results indicate that the ATP-induced elevation of Müller glial DNA synthesis is dependent on an influx of calcium ions from the extracellular space and that the inhibiting effect of BK channel blockers on ATP-evoked DNA synthesis is caused by an inhibition of this influx. The amount of the calcium influx seems to be directly correlated to the strength of the ATP-evoked proliferation.

Adenosine Triphosphate↗

PubMed2002

Upregulation of extracellular ATP-induced Müller cell responses in a dispase model of proliferative vitreoretinopathy.

PURPOSE: To test whether in an animal model of proliferative vitreoretinopathy (PVR) the Müller glial cells displayed an upregulation of purinergic P2 receptor-mediated responses. METHODS: 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. RESULTS: 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. Further, significant PVR-induced increase was observed in the number of Müller cells responding to adenosine 5'-triphosphate (ATP), with a transient elevation of their [Ca(2+)](i). If isolated Müller cells were exposed to ATP, 13% of the control cells, but 29% (moderate PVR) or 53% (massive PVR) of the reactive cells, showed fluorometric Ca(2+) increases. An increase of Ca(2+)-activated K(+) currents was measured in 11% of the control cells, but in 83% (moderate PVR) and 90% (massive PVR) of the reactive cells. Confocal images of retinal wholemounts revealed similar results. Because similar responses were elicited by uridine triphosphate (UTP), the dominant involvement of metabotropic (P2Y type) purinergic receptors is suggested. CONCLUSIONS: An upregulation of purinergic receptors is part of the reactive changes of Müller cells during PVR. It is suggested that ATP-evoked Ca(2+) responses may support the proliferation of Müller cells during PVR.

Adenosine Triphosphate↗