Biomedical subjects

Andreas Reichenbach

Publications and source records attributed to Andreas Reichenbach.

81 records · Page 5Linked to original sources

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↗

PubMedDate not supplied

Endothelin receptors in the detached retina of the pig.

Endothelin-1 (ET-1) is a potent vasoconstrictor that causes hypoperfusion of the neurosensory retina. We investigated immunohistochemically the expression of the receptors for ET-1, ET(A) and ET(B), in control and locally detached retinas of the pig. Immunoreactivity for ET(A) was expressed in the innermost retinal layers and in the outer plexiform layer in control retinas, and was additionally strongly expressed by retinal blood vessels at 7 days after detachment of the sensory retina from the pigment epithelium. Immunoreactivity for ET(B) was expressed by the innermost retinal layers, by ganglion cell somata, and by Müller glial cells in the control tissue, and was not altered in its expression after detachment. The vascular expression of ET(A) may suggest a hypoperfusion of the retina after detachment.

Animals↗

PubMedDate not supplied

Retinal endothelial angiogenic activity: effects of hypoxia and glial (Müller) cells.

OBJECTIVE: To explore the impact of retinal glial (Müller) cells on survival and neovascularization-related activities of cultured retinal endothelial cells under normoxic and hypoxic conditions. METHODS: Bovine retinal endothelial cells (BRECs) were cultured under normoxia or hypoxia (0.5% O2) either alone, together with the human Müller cell line MIO-M1, or in normoxia- or hypoxia-conditioned media of MIO-M1 cells. Cell number, proliferation, apoptotic cell death, and migration of BRECs were determined. RESULTS: Exposure of BRECs to hypoxia for 24 h decreased the number of adherent cells and the proliferation rate, but increased apoptosis and cell migration. Increased apoptosis and decreased proliferation of the BRECs occurred also in the presence of conditioned media of MIO-M1 cells. Under normoxic conditions, co-culture with MIO-M1 cells resulted in increased proliferation, but decreased apoptosis and migration rates of BRECs. Under hypoxic conditions, the Müller cells released elevated amounts of VEGF but their presence decreased proliferation, apoptosis and the migration rates of BRECs. CONCLUSIONS: Hypoxia inhibits the proliferation of retinal endothelial cells. Müller cells release soluble mediators that enhance this hypoxia-mediated effect but, under certain conditions (i.e., in co-culture), may protect retinal endothelial cells from apoptosis, thus supporting their survival. Altogether the findings indicate that the key signal necessary to trigger retinal endothelial proliferation under hypoxia remains to be determined.

Animals↗

PubMedDate not supplied

Pathomechanisms of cystoid macular edema.

Cystoid macular edema (CME) is a well-known endpoint of various ocular diseases, but the relative pathogenic impact of extra- and intracellular fluid accumulation within the retinal tissue still remains uncertain. While most authors favor an extracellular fluid accumulation as the main causative factor of cyst formation, there are indications that Müller cell swelling may also contribute to CME development (particularly in cases without significant angiographic vascular leakage). Vascular leakage occurs after a breakdown of the blood-retinal barrier during traumatic, vascular, and inflammatory ocular diseases, and allows the serum to get into the retinal interstitium. Since intraretinal fluid distribution is restricted by two diffusion barriers, the inner and outer plexiform layers, serum leakage from intraretinal vessels causes cysts mainly in the inner nuclear layer while leakage from choroid/pigment epithelium generates (in addition to subretinal fluid accumulation) cyst formation in the Henle fiber layer. In the normal healthy retina, the transretinal water fluxes are mediated by glial and pigment epithelial cells. These water fluxes are inevitably coupled to fluxes of osmolytes; in the case of glial (Müller) cells, to K(+) clearance currents. For this purpose, the cells express a complex, microtopographically optimized pattern of transporters and channels for osmolytes and water in their plasma membrane. Ischemic/hypoxic alterations of the retinal microvasculature result in gliotic responses which involve down-regulation of K(+) channels in the perivascular Müller cell end-feet. This means a closure of the main pathway which normally generates the osmotic drive for the redistribution of water from the inner retina into the blood. The result is an intracellular K(+) accumulation which, then, osmotically drives water from the blood into the glial cells (i.e., in the opposite direction) and causes glial cell swelling, edema, and cyst formation. While the underlying mechanisms await further research, it is expected that their improved knowledge will stimulate the development of novel therapeutic approaches to resolve edema in retinal tissue.

Blood-Retinal Barrier↗