Winfried Reichelt

Role of glial K(+) channels in ontogeny and gliosis: a hypothesis based upon studies on Müller cells.

The electrophysiological properties of Müller cells, the principal glial cells of the retina, are determined by several types of K+ conductances. Both the absolute and the relative activities of the individual types of K+ channels undergo important changes in the course of ontogenetic development and during gliosis. Although immature Müller cells express inwardly rectifying K+ (KIR) currents at a very low density, the membrane of normal mature Müller cells is predominated by the KIR conductance. The KIR channels mediate spatial buffering K+ currents and maintain a stable hyperpolarized membrane potential necessary for various glial‐neuronal interactions. During “conservative” (i.e., non‐proliferative) reactive gliosis, the KIR conductance of Müller cells is moderately reduced and the cell membrane is slightly depolarized; however, when gliotic Müller cells become proliferative, their KIR conductances are dramatically down‐regulated; this is accompanied by an increased activity of Ca2+‐activated K+ channels and by a conspicuous unstability of their membrane potential. The resultant variations of the membrane potential may increase the activity of depolarization‐activated K+, Na+ and Ca2+ channels. It is concluded that in respect to their K+ current pattern, mature Müller cells pass through a process of dedifferentiation before proliferative activity is initiated. GLIA 29:35–44, 2000.

Loss of inwardly rectifying potassium currents by human retinal glial cells in diseases of the eye

We compared the inward K+ currents of Müller glial cells from healthy and pathologically changed human retinas. To this purpose, the whole‐cell voltage‐clamp technique was performed on noncultured Müller cells acutely isolated from human retinas. Cells originated from retinas of four healthy organ donors and of 24 patients suffering from different vitreoretinal and chorioretinal diseases. Müller cells from organ donors displayed inward K+ currents in the whole‐cell mode similar to those found in other species. In contrast, this pattern was clearly changed in the Müller cells from patient retinas. In whole‐cell recordings many Müller cells had strongly decreased inward K+ current amplitudes or lost these currents completely. Thus, the mean input resistance of Müller cells from patients was significantly increased to 1,129 ± 812 MΩ, compared to 279 ± 174 MΩ in Müller cells from healthy organ donor retinas. Accordingly, since the membrane potential is mainly determined by the K+ inward conductance in healthy Müller cells, a large amount of Müller cells from patient retinas had a membrane potential which was significantly lower than that of Müller cells from control eyes. The mean membrane potentials were −37 ± 24 mV and −63 ± 25 mV for patient and donor Müller cells, respectively. The newly described membrane characteristic changes of Müller cells from patient eyes are assumed to interfere severely with normal retinal function: (1) the retinal K+ homeostasis, which is partly regulated by the Müller cell‐mediated spatial buffering, should be disturbed, and (2) the diminished membrane potential should influence voltage‐dependent transporter systems of the Müller cells, e.g., the Na+‐dependent glutamate uptake. GLIA 20:210–218, 1997.

The glutathione level of retinal Müller glial cells is dependent on the high-affinity sodium-dependent uptake of glutamate

The dependence of intracellular glutathione, an important radical scavenger, on the extracellular glutamate and cystine concentration and the velocity of the high affinity sodium/glutamate transporter was studied in freshly-isolated Müller glial cells of the guinea-pig, kept in vitro for up to 11 h. To this end the relative Müller cell glutathione levels were measured using the fluorescent dye monochlorobimane, using different concentrations of glutamate and cystine in Ringer solution. In some experiments L-buthionine-[S,R]-sulfoximine, a blocker of glutathione synthesis, or L-trans-pyrrolidine-2,4-dicarboxylic acid and L-alpha-aminoadipic acid, inhibitors of glutamate uptake, were added. The Müller cells maintained about 80% of the normal glutathione level when maintained in Ringer solution containing 100 microM glutamate for 11 h. When under these conditions 100 microM cystine was added, the glutathione level increased to values, which were even higher than those at the beginning of the incubation period. Addition of cystine without glutamate caused a run down of the glutathione level to about 45% of the normal level, which is comparable to the run down in pure Ringer solution. Likewise, application of L-buthionine-[S,R]-sulfoximine (5 mM) lead to a strong run down of the glutathione level even in glutamate/cystine (100 microM)-containing solution. A similar suppressing effect was observed using L-trans-pyrrolidine-2,4-dicarboxylic acid and L-alpha-aminoadipic acid in the presence of 100 microM cystine and glutamate. We conclude that the intracellular glutamate concentration of the Müller cells is determined by the extracellular glutamate concentration and the velocity of the sodium/glutamate uptake. Consequently, cystine uptake into Müller cells, which is performed by the cystine/glutamate antiporter, is fueled by the sodium/glutamate transporter with intracellular glutamate. Both glutamate and cystine are also substrates for glutathione synthesis. The glutathione level is logically limited by the capacity of the sodium/glutamate transporter to provide glutamate intracellularly for, first, cystine uptake and, second, direct insertion into glutathione. Accordingly, the glutathione level is reduced when the sodium/glutamate transporter is blocked. Thus, a diminution of the glutathione level should be taken into consideration when the effects of sodium/glutamate uptake failure and reduced intracellular glutamate concentrations are discussed.

Ionic mechanisms in glutamate‐induced astrocyte swelling: Role of K+ influx

L‐Glutamate (L‐GLU) induced astrocyte swelling in a time‐ and concentration‐dependent, as well as Na+‐ and Ca2+‐dependent, and Cl‐‐independent manner. Swelling was prevented by MK‐801, cystine, and ouabain. Since L‐GLU swelling is ionically dependent, we determined the role of various ions in such swelling. Our results indicate that K+ uptake plays a major role in the mechanism of L‐GLU‐induced astrocyte swelling. Like swelling, K+ uptake is dependent on Ca2+ and Na+, but not on Cl‐. Likewise, K+ uptake was inhibited by MK‐801, cystine, and ouabain. The K+ channel blockers, Ba2+ and tetraethylammonium, partially prevented L‐GLU‐induced swelling. In addition to K+ channels, K+ influx may also be mediated through Na+/K+‐ATPase, as its activity is increased by L‐GLU uptake along with the required Na+. Taken together, the data suggest that K+ influx plays a key role in the mechanism of L‐GLU‐mediated astrocyte swelling. J. Neurosci. Res. 52:307–321, 1998.

Characterization of cystine uptake in cultured astrocytes

Glutathione is involved in the maintenance of the structural and functional integrity of membrane proteins, in protection against free radicals and oxidative stress, and in the detoxification of xenobiotics. The cellular uptake of cystine is the rate limiting step in the biosynthesis of glutathione. The precise mechanism for such uptake is not clear as some reports indicate that the uptake occurs through a glutamate-cystine antiporter (system X(c)(-)), whereas, others suggest that it is taken up by the glutamate transporter (system X(AG)). Our studies in cultured astrocytes derived from neonatal rats showed that glutamate, D- and L-aspartate inhibited cystine uptake; that factors that increased intracellular glutamate levels, which would have enhanced the activity of the antiporter, did not stimulate cystine uptake; that the uptake was sodium dependent and partially chloride dependent; that the b(o,+) and ASC systems, which have been shown to carry cystine in some cells, did not mediate cystine uptake in astrocytes; that glutamate uptake blockers such as L-aspartate-beta-hydroxamate (AbetaH) and L-trans-pyrrolidine-2,4-dicarboxylate (PDC), as well as cystine uptake inhibitor L-alpha-aminoadipate (AAA) potently reduced cystine uptake. Additionally, deferoxamine (100 microM) as well as ammonium chloride (5 mM), both of which inhibit glutamate uptake, also inhibited cystine uptake. Taken together, our findings indicate that astrocytes take up cystine through a similar, if not identical, system used to take up glutamate. Interference of cystine uptake by astrocytes through the glutamate transport system may have profound effects on the redox state and the structural and functional integrity of the CNS.

Three distinct types of voltage-dependent K+ channels are expressed by Müller (glial) cells of the rabbit retina

There is ample evidence that retinal radial glial (Müller) cells play a crucial role in retinal ion homeostasis. Nevertheless, data on the particular types of ion channels mediating this function are very rare and incomplete; this holds especially for mammalian Müller cells. Thus, the whole-cell variation of the patch-clamp technique was used to study voltage-dependent currents in Müller cells from adult rabbit retinae. The membrane of Müller cells was almost exclusively permeable to K+ ions, as no significant currents could be evoked in K+-free internal and external solutions, external Ba2+ (1 mM) reversibly blocked most membrane currents, and external Cs+ ions (5 mM) blocked all inward currents. All cells expressed inwardly rectifying channels that showed inactivation at strong hyperpolarizing voltages (≥ −120 mV), and the conductance of which varied with the square root of extracellular K+ concentration ([K+]e). Most cells responded to depolarizing voltages (≥ −30 mV) with slowly activating outward currents through delayed rectifier channels. These currents were reversibly blocked by external application of 4-aminopyridine (4-AP, 0.5 mM) or tetraethylammonium (TEA, > 20 mM). Additionally, almost all cells showed rapidly inactivating currents in response to depolarizing (≥ −60 mV) voltage steps. The currents were blocked by Ba2+ (1 mM), and their amplitude increased with the [K+]e. Obviously, these currents belonged to the A-type family of K+ channels. Some of the observed types of K+ channels may contribute to retinal K+ clearance but at least some of them may also be involved in regulation of proliferative activity of the cells.

Comparative studies on mammalian Muller (retinal glial) cells

Muller cells from 22 mammalian species were subjected to morphological and electrophysiological studies. In the ‘mid-periphery’ of retinae immunocytochemically labeled for vimentin, estimates of Muller cell densities per unit retinal surface area, and of neuron-to-(Muller) glia indices were performed. Muller cell densities were strikingly similar among the species studied (around 8000–11000 mm−2) with the extremes of the horse (≤5000 mm−2) and the tree shrew (≥20000 mm−2). By contrast, the number of neurons per Muller cell varied widely, being clustered at 6–8 (in retinae with many cones), at about 16, and at up to more than 30 (in strongly rod-dominated retinae). Isolated Muller cell volumes were estimated morphometrically, and cell surface areas were calculated from membrane capacities. Muller cells isolated from thick vascularized retinae (carnivores,rats, mice, ungulates) were longer and thinner, and had smaller volumes but higher surface-to-volume ratios than cells from thin paurangiotic (i.e. with blood vessels only near the optic disc) or avascular retinae (rabbits, guinea pigs, horses, zebras). In whole-cell voltage-clamp studies, Muller cells from all mammals studied displayed two dominant K+ conductances, inwardly rectifying currents and delayed rectifier currents. TTX-sensitive Na+ currents were recorded only in some species. Based on these data, the following hypotheses are presented, (a) neuron-to-(Muller) glia indices are determined by precursor cell proliferation rather than by metabolic demands; (b) Muller cell volumes depend on available space rather than on the number of supported neurons; and (c) it follows that, the specific metabolic activities of Muller cells must differ greatly between species, a difference that may contribute to distinct patterns of retinal vascularization.

Voltage-dependent K+ currents in guinea pig Müller (glial) cells show different sensitivities to blockade by Ba2+

The effect of externally applied Ba2+ and Na+ on K+ currents was investigated by means of whole-cell patch-clamp in isolated and in situ Müller cells from guinea pig retina. Müller cells express a typical set of K+ currents, i.e. an ohmic current, an inactivating inward current (IK(IR)), a delayed rectifier (IK(DR)) and an inactivating outward current (IK(A)). Inactivation of the inward current did not occur when extracellular Na+ was replaced by choline. When administered in increasing concentrations, Ba2+ blocked these K+ currents in a typical sequence: the ohmic current and IK(A) were most sensitive, followed by IK(IR), whereas IK(DR) was not completely blocked even in 1 mM Ba2+. The differential sensitivity of Müller cell K+ currents to external Ba2+ may be a tool which can be used to improve our understanding of the Müller cell response to physiological stimulation of the retina.

Experimental retinal detachment causes widespread and multilayered degeneration in rabbit retina

Retinal detachment remains one of the most frequent causes of visual impairment in humans, even after ophthalmoscopically successful retinal reattachment. This study was aimed at monitoring (ultra-) structural alterations of retinae of rabbits after experimental detachment. A surgical procedure was used to produce local retinal detachments in rabbit eyes similar to the typical lesions in human patients. At various periods after detachment, the detached retinal area as well as neighbouring attached regions were studied by light and electron microscopy. In addition to the well-known degeneration of photoreceptor cells in the detached retina, the following progressive alterations were observed, (i) in both the detached and the attached regions, an incomplete but severe loss of ganglion cell axons occurs; (ii) there is considerable ganglion cell death, particularly in the detached area; (iii) even in the attached retina distant from the detachment, small adherent groups of photoreceptor cells degenerate; (iv) these photoreceptor cells degenerate in an atypical sequence, with severely destructed somata and inner segments but well-maintained outer segments; and (v) the severe loss of retinal neurons is not accompanied by any significant loss of Müller (glial) cells. It is noteworthy that the described progressive (and probably irreparable) retinal destructions occur also in the attached retina, and may account for visual impairment in strikingly large areas of the visual field, even after retinal reattachment.

GABA uptake into isolated retinal Müller glial cells of the guinea-pig detected electrophysiologically

In order to investigate GABA uptake into Müller glial cells electrophysiologically, we used the whole-cell voltage-clamp technique. Since Müller cells were insensitive to application of muscimol and baclofen, the expression of GABAA and GABAB receptors can be excluded. Therefore, the observed GABA current must be due to an electrogenic GABA transporter. This transporter is driven by the transmembrane Na+ gradient, as replacement of the extracellular Na+ by choline inhibits the GABA current. Currents evoked by cis-4-aminocrotonic acid, a GABAC specific agonist, are also inhibited by replacement of extracellular Na+, indicating that this compound is a substrate for the uptake. The competitive GABA uptake blockers beta-alanine and nipecotic acid are substrates of the transporter as well, and produce 42% and 65% of the currents elicited by the same GABA concentration, respectively. Affinity of the transporter for GABA is high, the Km value being 5 microM at -80 mV.