Role of taurine in osmoregulation in brain cells: Mechanisms and functional implications

Summary All cells including neurons and glial cells are able to keep their volume within a very limited range. The volume regulatory mechanism involves changes in the concentration of osmolytes of which taurine appears to be of particular importance in brain cells. Swelling in brain cells may occur as a result of depolarization or small fluctuations in osmolarity. In isolated brain cells these conditions will always lead to a release of taurine, the time course of which is superimposable on that of the volume regulatory decrease which follows the initial cell swelling. The mechanism responsible for taurine release associated with swelling has not been fully elucidated but a large body of evidence seems to exclude participation of the taurme high affinity carrier. Using a number of inhibitors of anion exchangers it has been demonstrated that both volume regulation and taurine release in brain cells are inhibited by these drugs, implicating an anion channel in the process. It has be controversial issue as to whether or not taurine release is Ca++ dependent. Recent evidence appears to suggest that the release process is not associated with Ca⁺⁺ or Ca⁺⁺ channels. It is, however, quite possible that the swelling process may involve the Ca⁺⁺ calmodulin system or other second messengers. Taurine also contributes to volume regulation after shrinkage of brain cells, in this case by increasing its intracellular concentration. This change is accomplished byan upregulation of the Na+/taurine cotransporter, together with reduced passive fluxes and increased endogenous synthesis.     

Distribution of mitochondria within Muller cells – I. Correlation with retinal vascularization in different mammalian species

The distribution of mitochondria within retinal glial (Muller) cells and neurons was studied by electron microscopy, by confocal microscopy of a mitochondrial dye and by immunocytochemical demonstration of the mitochondrial enzyme GABA transaminase (GABA-T). We studied sections and enzymatically dissociated cells from adult vascularized (human, pig and rat) and avascular or pseudangiotic (guinea-pig and rabbit) mammalian retinae. The following main observations were made. (1) Muller cells in adult euangiotic (totally vascularized) retinae contain mitochondria throughout their length. (2) Muller cells from the periphery of avascular retinae display mitochondria only within the sclerad-most end of Muller cell processes. (3) Muller cells from the vascularized retinal rim around the optic nerve head in guinea-pigs contain mitochondria throughout their length. (4) Muller cells from the peripapillar myelinated region (‘medullary rays’) of the pseudangiotic rabbit retina contain mitochondria up to their soma. In living dissociated Muller cells from guinea-pig retina, there was no indication of low intracellular pH where the mitochondria were clustered. These data support the hypothesis that Muller cells display mitochondria only at locations of their cytoplasm where the local O2 pressure (pO2) exceeds a certain threshold. In contrast, retinal ganglion cells of guinea-pig and rabbit retinae display many mitochondria although the local pO2 in the inner (vitread) retinal layers has been reported to be extremely low. It is probable that the alignment of mitochondria and the expression of mitochondrial enzymes are regulated by different mechanisms in various types of retinal neurons and glial cells.     

EFFECTS OF POTASSIUM ON INDICATOR SPACES AND FLUXES IN SLICES OF BRAIN CORTEX FROM ADULT AND NEW-BORN RATS

—Inulin, sucrose and chloride spaces were measured in slices of brain cortex from adult and from new-born rats incubated in‘balanced', potassium-rich and sodium-rich media. The efflux of the radioactive markers was followed in the two first media and the following results were obtained: (1) In brain slices from new-born rats inulin and sucrose spaces were of identical magnitude (35 per cent). The space magnitude was essentially unaffected by excess potassium. The chloride space was somewhat larger than the inulin (sucrose) space, and the difference increased continuously but relatively slightly with the external potassium concentration. By far the largest amount (i.e. about 90 per cent) of the efflux of radioactive inulin, sucrose and chloride occurred from a rapidly exchanging compartment during incubation in both ‘balanced’ and potassium-rich media. (2) In brain slices from adult rats the inulin space (35 per cent) was significantly smaller than that of sucrose (50 per cent) and of chloride (65 per cent); it seemed to represent the extracellular space relatively well although 10 per cent of the efflux occurred from a slowly exchanging (probably intracellular) compartment. High concentrations of potassium led to a reduction of the inulin space which was probably a result of the concomitant intracellular swelling. The hyperosmolarity per se did not affect the space magnitude, but an increase of the sodium concentration exerted a competitive inhibition of potassium effects on the inulin space. Of the sucrose efflux, 20 per cent occurred from a slowly exchanging compartment in both ‘balanced’ and potassium-rich media, and 30 per cent of the chloride exchanged with this compartment when the tissue was incubated in a ‘balanced’ medium. An increase of the external potassium concentration caused a drastic increase of the chloride space and a reduction of the slowly exhanging fraction of chloride efflux to less than 10 per cent.     

Astrocytic Metabolism Focusing on Glutamate Homeostasis: A Short Review Dedicated to Vittorio Gallo

A large number of studies have during the last several decades shown that astrocytes play a significant role in brain energy metabolism accounting for a considerable part of the oxygen uptake and the corresponding oxidative metabolism of glucose and lactate. Interestingly, it has become clear that in addition to these two major energy substrates, glutamate may be considered as an important alternative energy substrate and this is tightly coupled to its role as an excitatory neurotransmitter. Hence, this short review will link these events and provide an account of the role that Vittorio Gallo came to play as he coauthored a publication which demonstrated the usefulness of cultured cerebellar granule cells for studies of glutamate neurotransmission. Just by chance this study was published the same year that my own group published a similar study of glutamate uptake and release in a corresponding preparation of cultured neurons and astrocytes from cerebellum and cerebral cortex. Thus, it is a pleasure to dedicate this account of the role of astrocytes in glutamate neurotransmission to Vittorio Gallo whom I have had the pleasure of knowing for more than three decades.

     

Factor XII binding to endothelial cells depends on caveolae.

It is now generally accepted that factor XII (FXII) binds to cellular surfaces in the vascular system. One of the suggested receptors of this binding is the glycosylphosphatidylinositol-anchored urokinase-like plasminogen activator (u-PAR) harbored in caveolae/lipid rafts. However, binding of FXII to human umbilical vein endothelial cells (HUVEC) has never been shown to be localized to these specialized membrane structures. Using microscopical techniques, we here report that FXII binds to specific patches of the HUVEC plasma membrane with a high density of caveolae. Further investigations of FXII binding to caveolae were performed by sucrose density-gradient centrifugations. This showed that the majority of FXII, chemically cross-linked to HUVEC, could be identified in the same fractions of the gradient as caveolin-1, a marker of caveolae, while the majority of u-PAR was identified in noncaveolae lipid rafts. Accordingly, cholesterol-depleted cells were found to bind significantly reduced amounts of FXII. These observations, combined with the presence of a minority of u-PAR in caveolae concomitant with FXII binding, indicate that FXII binding to u-PAR may be secondary and depends upon the structural elements within caveolae. Thus, FXII binding to HUVEC depends on intact caveolae on the cellular surface.     

Glycogen Shunt Activity and Glycolytic Supercompensation in Astrocytes May Be Distinctly Mediated via the Muscle Form of Glycogen Phosphorylase

Glycogen is the main storage form of glucose in the brain. In contrast with previous beliefs, brain glycogen has recently been shown to play important roles in several brain functions. A fraction of metabolized glucose molecules are being shunted through glycogen before reentering the glycolytic pathway, a phenomenon known as the glycogen shunt. The significance of glycogen in astrocyte energetics is underlined by high activity of the glycogen shunt and the finding that inhibition of glycogen degradation, under some conditions leads to a disproportional increase in glycolytic activity, so-called glycolytic supercompensation. Glycogen phosphorylase, the key enzyme in glycogen degradation, is expressed in two different isoforms in brain, the muscle and the brain isoform. Recent studies have illustrated how these are differently regulated. In the present study, we investigate the role of the two isoforms in glycolytic supercompensation in cultured astrocytes with the expression of either one of the isoforms silenced by siRNA knockdown. When reintroducing glucose to glucose-starved astrocytes, glycolytic activity increased dramatically. Interestingly, the increase was 30% higher in astrocytes not expressing the muscle isoform of glycogen phosphorylase. Based on these results and previously published data we couple the muscle isoform of glycogen phosphorylase to glycolytic supercompensation and glycogen shunt activity, giving insights to the underlying mechanistic of these phenomena.     

The structure of a mixed GluR2 ligand-binding core dimer in complex with (S)-glutamate and the antagonist (S)-NS1209

Ionotropic glutamate receptors (iGluRs) mediate fast synaptic transmission between cells of the central nervous system and are involved in various aspects of normal brain function. iGluRs are implicated in several brain disorders, e.g. in the high-frequency discharge of impulses during an epileptic seizure. (RS)-NS1209 functions as a competitive antagonist at 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionate receptors, and shows robust preclinical anticonvulsant and neuroprotective effects. This study explores 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionate receptor binding and selectivity of this novel class of antagonists. We present here the first X-ray structure of a mixed GluR2 ligand-binding core dimer, with the high-affinity antagonist (S)-8-methyl-5-(4-(N,N-dimethylsulfamoyl)phenyl)-6,7,8,9,-tetrahydro-1H-pyrrolo[3,2-h]-isoquinoline-2,3-dione-3-O-(4-hydroxybutyrate-2-yl)oxime [(S)-NS1209] in one protomer and the endogenous ligand (S)-glutamate in the other. (S)-NS1209 stabilises an even more open conformation of the D1 and D2 domains of the ligand-binding core than that of the apo structure due to steric hindrance. This is the first time ligand-induced hyperextension of the binding domains has been observed. (S)-NS1209 adopts a novel binding mode, including hydrogen bonding to Tyr450 and Gly451 of D1. Parts of (S)-NS1209 occupy new areas of the GluR2 ligand-binding cleft, and bind near residues that are not conserved among receptor subtypes. The affinities of (RS)-NS1209 at the GluR2 ligand-binding core as well as at GluR1-6 and mutated GluR1 and GluR3 receptors have been measured. Two distinct binding affinities were observed at the GluR3 and GluR4 receptors. In a functional in vitro assay, no difference in potency was observed between GluR2(Q)(o) and GluR3(o) receptors. The thermodynamics of binding of the antagonists (S)-NS1209, DNQX and (S)-ATPO to the GluR2 ligand-binding core have been determined by displacement isothermal titration calorimetry. The displacement of (S)-glutamate by all antagonists was shown to be driven by enthalpy.     

Evidence for net uptake of GABA into mouse astrocytes in primary cultures—Its sodium dependence and potassium independence

Content of GABA was measured in cultured, normal astrocytes (from the brain cortex of newborn mice) together with the effect of nonradioactive GABA on the efflux of labeled GABA from cells previously loaded with [¹⁴C]GABA. An increase of external GABA concentration from 0 to 25 M evoked a rise of the GABA content in the cells to a level which was approximately 50 times that of the incubation medium. Neither 200 nor 2000 M nonradioactive GABA had any effect on the rate of release of radioactivity from cells loaded with [¹⁴C]GABA. Both the high tissue/medium ratio and the lack of a GABA-induced enhancement of the release of radioactivity indicate that the previously observed high-affinity uptake of GABA in cultured astrocytes represents a net uptake and not a homoexchange with endogenous GABA. This uptake is sodium dependent but was found to be unaffected in potassium-free media; the quantitative correlation between GABA transport and sodium transport differed from that reported for synaptosomes.     

Observations on a tissue response within the body cavity of fish infected with the plerocercoid larvae of Ligula intestinalis (L.) (Cestoda: Pseudophyllidea)

Infection of certain species of cyprinid fish with the plerocercoid larva of the cestode Ligula intestinalis is accompanied by a host tissue response.
The tissue reaction is evident in roach Rutilus rutilus as young as three months. An initial cellular infiltration is followed by the laying down of sheets of connective tissue. A case of calcification is described in a single infected rudd Scardinius erythrophthalmus .