Harold K. Kimelberg

Swelling-induced release of glutamate, aspartate, and taurine from astrocyte cultures

Swelling of primary astrocyte cultures by exposing them to hypotonic media caused release of label after the cells had been allowed to accumulate 3H-L-glutamate, 3H-D-aspartate, or 3H-taurine. Comparable release of endogenous L-glutamate or taurine, as measured by high- pressure liquid chromatography (HPLC), was also found. Release of label was not affected by treating the cells with cytochalasin B, indicating that microfilament polymerization was not significantly involved. Hypotonic-induced release did not appear to principally involve reversal of the Na(+)-dependent uptake system since increasing external K+ to depolarize the cells by replacement of external Na+, thus maintaining isotonic conditions, increased release to a lesser extent. Threo beta-hydroxyaspartate, a potent 3H-L-glutamate uptake blocker, added externally stimulated efflux of 3H-L-glutamate independently of the swelling-induced efflux. Upon restoration of swollen cells to isotonic medium they showed an unimpaired ability to take up 3H-L- glutamate. The swelling-induced release of label was inhibited by a number of anion transport inhibitors, one of which has been shown to significantly improve outcome in an experimental brain trauma/hypoxia model in which astrocyte swelling is an early event.

Phospholipid requirements for (Na+ + K+)-ATPase activity: Head-group specificity and fatty acid fluidity☆

We have re-examined the question of phospholipid activation of latent (Na+ + K+)-ATPase activity in delipidized preparations. The influence of both polar head-groups and fatty acyl chain fluidity have been examined. The results indicate the following: 1. 1. The enzyme can be reactivated not only by phosphatidylserine, but also equally well by phosphatidylglycerol. A large variety of other purified phospholipids were found to have little or no effect. 2. 2. This specificity for (Na+ + K+)-ATPase reactivation correlates with the observation that only phosphatidylserine and phosphatidylglycerol vesicles show substantial discrimination for K+ over Na+ in terms of permeability. 3. 3. Maximal reactivation is obtained only when the fatty acyl chains are fluid. Activation by dipalmitoyl phosphatidylglycerol is inhibited below its transition temperature, or in the presence of cholesterol. Arrhenius plots for the activation by dipalmitoyl phosphatidylglycerol show a break for (Na+ + K+-ATPase activity corresponding to the presumed transition temperature for the gel to liquid-crystalline transition for this phospholipid. We suggest the term “viscotropic” to describe such effects of membrane fluidity on enzyme activity. 4. 4. The ATPase preparations used in this paper were obtained by deoxycholate treatment of microsomal fractions of rabbit kidney and beef brain. This treatment results in the removal of more than 90% of the phospholipid and inhibition of (Na+ + K+-ATPase activity. It was noted that exclusive specificity for reactivation by phosphatidylserine and phosphatidylglycerol was obtained only with those preparations where the inhibition of (Na+ + K+)-ATPase activity was essentially complete. 5. 5. These results are discussed in relation to previous work on the phospholipid requirement for (Na+ + K+)-ATPase activity, the ability of phospholipids to exhibit K+Na+ discrimination and current concepts of protein-lipid interactions in biological membranes.

Role of cholesterol in membranes effects on phospholipid-protein interactions, membrane permeability and enzymatic activity

Abstract The effect of cholesterol on the interaction of proteins with phospholipid membranes was studied using three independent techniques: effects on vesicle permeability, monolayer expansion, and phospholipid-dependent (Na+ + K+)-ATPase activity. The proteins studied were: cytochrome c, albumin, hemoglobin, lysozyme, myelin basic protein, myelin proteolipid apoprotein, and the polypeptide gramicidin A. The results were as follows: 1. 1. All the proteins in this study produced a large increase in the permeability of phospholipid vesicles to Na+. When cholesterol was mixed with phospholipid in equimolar proportions, most proteins produced only a comparatively small increase in permeability. This inhibitory effect of cholesterol on the permeability of phospholipid-protein membranes, was 38-fold for cytochrome c and 10-fold for hemoglobin. The only protein that was not affected by cholesterol was the myelin proteolipid apoprotein. Experiments with cytochrome c indicated that the above effects were unlikely to be due to inhibition of its binding to phosphatidylserine vesicles. 2. 2. The presence of cholesterol in phosphatidylserine monolayers inhibits the area expansion produced by the addition of cytochrome c to the bulk phase. The inhibition of monolayer expansion by cholesterol was shown to be considerably larger than that obtained by equivalent dilution of the surface charges of phosphatidylserine by phosphatidylcholine. 3. 3. The presence of cholesterol inhibits the ability of phospholipids to activate a delipidated preparation of (Na+ + K+)-ATPase. The degree of inhibition produced by nearly equimolar mixtures of cholesterol with brain phosphatidylserine and dioleylphosphoatidylglycerol was approximately 60% while in mixtures with dipalmitoylphosphatidylglycerol, it was more than 90%. 4. 4. The biological significance of these data are discussed in relation to the possible effects of increased cholesterol levels in cell membranes during the process of aging and the development of atherosclerosis.

Astrocytic swelling in cerebral ischemia as a possible cause of injury and target for therapy.

In this viewpoint article, I summarize data showing that the astrocytic swelling that occurs early after the acute CNS pathologies ischemia and traumatic brain injury is damaging. We have proposed that one reason may be the release of excitatory amino acids (EAA) via volume‐activated anion channels (VRACs) that are activated by such swelling. This release could be a target for therapy, which could involve blocking the astrocytic swelling or the release mechanisms. The transport mechanisms likely causing the early astrocytic swelling are therefore summarized. In terms of targeting the release mechanisms, we have found a potent inhibitor of VRACs, tamoxifen, to be strongly neuroprotective in focal ischemia with a therapeutic window of 3 h after initiation of the ischemia. The question, however, of whether neuroprotection by tamoxifen can be solely attributed to VRAC inhibition in astrocytes has yet to be resolved. ©2005 Wiley‐Liss, Inc.

Furosemide- and bumetanide-sensitive ion transport and volume control in primary astrocyte cultures from rat brain

K+ and Cl- transport using 42K+ and 36Cl- was studied in primary astrocyte cultures prepared from neonatal rat brains. A component of 42K+ uptake was sensitive to both furosemide and bumetanide with maximum inhibition being obtained at 1 and 0.01 mM concentrations of the inhibitors, respectively. Furosemide and bumetanide also markedly inhibited uptake of 36Cl-. 42K+ uptake in the presence of ouabain was also sensitive to the omission of medium Na+ and Cl-. These results suggest the existence of a K+ + Na+ + Cl- cotransport system in astrocyte cultures which in many cells has been shown to be involved in volume regulation. We studied volume changes using uptake of [14C]3-O- methyl-D-glucose ([14C]3-OMG), and also ion transport, in attached cells in response to exposure to hyper- or hypotonic medium. Exposure to medium made hypertonic with mannitol resulted in shrinkage of the [14C]3-OMG space of the cells, but did not affect 36Cl- content, expressed as nmol/mg protein. Exposure to hypotonic medium led to a marked increase in the [14C]3-OMG space, rapidly followed by a decrease towards control values. After the cells were then exposed to isotonic medium there was an immediate decrease followed by a slower increase in the [14C]3-OMG space. The increase in the [14C]3-OMG space was partially inhibited by 1 mM furosemide.(ABSTRACT TRUNCATED AT 250 WORDS)

The problem of astrocyte identity

Astrocytes were the original neuroglia of Ramón y Cajal but after 100 years there is no satisfactory definition of what should comprise this class of cells. This essay takes a historical and philosophical approach to the question of astrocytic identity. The classic approach of identification by morphology and location are too limited to determine new members of the astrocyte population. I also critically evaluate the use of protein markers measured by immunoreactivity, as well as the newer technique of marking living cells by using promoters for these same proteins to drive reporter genes. These two latter approaches have yielded an expanded population of astrocytes with diverse functions, but also mark cells that traditionally would not be defined as astrocytes. Thus we need a combination of measures to define an astrocyte but it is not clear what this combination should be. The molecular approach, especially promoter driven fluorescent reporter genes, does have the advantage of pre marking living astrocytes for electrophysiological or imaging recordings. However, lack of sufficient understanding of the behavior of the inserted constructs has led to unclear results. This approach will no doubt be perfected with time but at present an acceptable, practical definition of what constitutes the class of astrocytes remains elusive.

EFFECTS OF NOREPINEPHRINE ON THE MORPHOLOGY AND SOME ENZYME ACTIVITIES OF PRIMARY MONOLAYER CULTURES FROM RAT BRAIN

Addition of norepinephrine or isoproterenol to primary cultures started from the brains of 1‐3 day old rats caused up to 200‐fold increases in cAMP levels, which reached a maximum by 5‐10 min and then declined. This effect was studied in detail for norepinephrine. The rise in cAMP levels was followed by morphological changes, in which up to 65% of the cells exhibited an astrocyte‐like morphology, and 2‐3 fold increases in carbonic anhydrase and (Na+‐K+) ATPase activities. However, morphological transformation also occurred after much smaller increases in total cAMP levels. These effects on cell morphology and enzyme activities reached a maximum 1‐2 h after addition of norepinephrine and then declined. Carbonic anhydrase activity was found both in the particulate and post 100,000 g supernatant fractions from homogenates of these cultured cells, and in the latter case the activity was activated 3‐fold by addition of cAMP. The significance of these obscrvations on the cellular localization of, and functional role for similar increases in cAMP in brain tissue is discussed.

Manganese Uptake and Efflux in Cultured Rat Astrocytes

Abstract: Astrocytes play a central role in manganese (Mn) regulation in the CNS. Using primary astrocyte cultures from neonatal rat brains, these studies demonstrate a specific highaffinity transport system for Mn2+. Saturation kinetics are clearly indicated by both l/s versus l/s plots (Km= 0.30 ± 0.03 μMM; Vmax= 0.30 ± 0.02 nmol/mg of protein/min) and plots of v versus [s]. Several divalent cations (Co2, Zn2+, and Pb2+) failed to inhibit the initial rate of 54Mn2+ uptake. In contrast, extracellular Ca2+ at 10 μM decreased 54Mn2+ uptake. Exchange with extracellular Mn2+ was not obligatory for the efflux of 54Mn2+ into extracellular medium because efflux occurred into Mn2+‐free extracellular medium, but efflux of 54Mn2+ was enhanced when astrocytes were equilibrated in the presence of unlabeled Mn2+. Efflux of 54Mn2+ was biphasic with both a rapid and a slow component. Efflux was most rapid during the first 10 min of incubation, with 27.5 ± 2.2% of 54Mn2+ transported extracellularly, and 37.2 ± 1.2% of preloaded 54Mn2+ was retained by the astrocytes at 120 min. These studies show, for the first time, that mammalian astrocytes can transport Mn via a specific transport system.

Dissociation of neonatal rat brain by dispase for preparation of primary astrocyte cultures

We describe the use of the neutral protease Dispase for the dissociation of neonatal rat brain tissue for the preparation of primary monolayer astrocyte cultures. The method involves 5 to 6 successive extractions with careful separation of sedimenting, undissociated tissue. This method gives an initial cell suspension of high viability (93.7±1.7% cells exclude trypan blue). In comparison trypsin (0.25%) dissociated tissue gave a cell suspension that showed a lower viability of 58.2±7.6%. Identical saturation densities of 1.1 to 1.2×104 cells/cm2 after two weeks in culture were obtained for a range of seeding densities from 1 to 4×104 cells/cm2 of the Dispase dissociated tissue. Staining for glial fibrillary acidic protein showed that 90–100% cells were positive for this astroglial marker. Thus, the use of Dispase for the initial dissociation of rat brain tissue seems to give primary astrocyte cultures which are very reproducible and homogeneous.

Primary astrocyte cultures—a key to astrocyte function

Summary1.Morphological studies have established the ubiquitous nature of astrocytes in the CNS. Their processes surround capillaries and synapses, form the subpial and subependymal layers, and seemingly invest every neuronal surface not covered by other neuronal surfaces or oligodendroglial membranes. Although such interrelationships have long suggested that astrocytes may play many critical roles, there still remains relatively little experimental information on the functions and properties of these cells.2.About a decade ago it became evident that primary cultures from neonatal rodent brains can consist predominantly of normal astrocytes. Based on these findings there is now an increasing number of studies in which such primary cultures are being used to help unravel the continuing enigma of the properties and functions of astrocytes. Aspects of this work are reviewed in this article. Such work has already shown that astrocytes in primary culture exhibit the basic electrophysiological characteristics which had been the only functional property well established for these cellsin situ.3.Further studies of the electrophysiological properties of these cells, which can be correlated with ion transport studies, are beginning to show that astrocytes may have more complex electrophysiological properties than had previously been supposed, as well as a number of important electrically silent ion fluxes. In addition, astrocytes in primary culture show uptake of and receptors for a number of transmitters, properties which have wide-ranging implications.4.Studies in culture also support workin vivo that astroglia may have an important role in neuronal development.