- Email: [email protected]
Altered taurine release following hypotonic stress in astrocytes from mice deficient for GFAP and vimentin
Molecular Brain Research 62 Ž1998. 77–81
Research report
Altered taurine release following hypotonic stress in astrocytes from mice deficient for GFAP and vimentin Mei Ding
a,1
, Camilla Eliasson a
b,1
, Christer Betsholtz b, Anders Hamberger a , Milos Pekny
b,)
Department of Anatomy and Cell Biology, UniÕersity of Goteborg, PO Box 420, SE-405 30 Goteborg, Sweden ¨ ¨ b Department of Medical Biochemistry, UniÕersity of Goteborg, PO Box 440, SE-405 30 Goteborg, Sweden ¨ ¨ Accepted 1 September 1998
Abstract Astrocytes maintain their volume in response to changes in osmotic pressure in their environment by an affluxrinflux of ions and organic osmoequivalents. The initial swelling of an astrocyte transferred to a hypoosmotic medium is thus reversed within minutes. The mechanisms which trigger this process as well as the sensors for cell volume are largely unknown, however, the cytoskeleton appears to be involved. We have addressed the role of one component of the cytoskeleton, the intermediate filaments, in the maintenance of astrocytic cell volume. Astrocytes from wild type mice were compared with cells from mice deficient for either glial fibrillary acidic protein ŽGFAP–r– . or vimentin Žvimentin–r– . and with astrocytes from mice deficient for both proteins ŽGFAP–r–vim–r– .. Whereas GFAP–r– and vimentin–r– cultured or reactive astrocytes retain intermediate filaments, the GFAP–r–vim–r– astrocytes are completely devoid of these structures. The rate of efflux of the preloaded osmoequivalent 3 H-taurine from primary and passaged cultures of astrocytes was monitored. A reduction of NaCl Ž25 mM. in the perfusion medium led to a 400–900% increase of 3 H-taurine afflux in astrocytes from wild type mice. The stimulated efflux was not significantly affected in astrocytes from GFAP–r– or vimentin–r– mice. However, the efflux from astrocytes from GFAP–r–vim–r– mice was 25–46% lower than the wild type levels. The results strengthen the role of the cytoskeleton in astrocyte volume regulation and suggest an involvement of intermediate filaments in the process. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Astrocyte; Transgenic mouse; GFAP; Vimentin; Taurine; RVD Žregulatory volume decrease.
1. Introduction Changes in volume and shape of astrocytes is a common feature of CNS pathology, i.e., after hypoxia, ischemia, trauma, sustained seizures or liver failure w13,18x. Such conditions also involve increased production of the intermediate filament ŽIF. proteins, glial fibrillary acidic protein ŽGFAP. and vimentin w10,14,28x. Chemical composition of IFs reflects developmental and cell-type specificity, however the exact functions of this part of the cytoskeleton remain largely unknown. The role of IFs in the maintenance of mechanical integrity of cells and tissues has been recently established, primarily based on studies of keratin deficiencies in humans and on studies on transgenic mice w11,12x. Neurofilaments, the IFs of nerve
) C orresponding author. Fax: q 46-31-416108; [email protected] 1 These authors contributed equally to this work.
E-m ail:
cells, were found to be crucial for the modulation of the calibre of axons w24x, while GFAP was proposed to play a role in the inductionrmaintenance of the blood–brain barrier w21,34x as well as in modulation of neuronal functions, such as long-term depression w37x and long-term potentiation w25x. Astrocytes in culture respond with rapid swelling even to moderately hypoosmotic media but return, in this environment, to their original volume in 10–15 min w2,4,15,16,17,30x. This process, known as regulatory volume decrease ŽRVD., is of physiological importance for many cell types and involves an efflux of intracellular osmoequivalents w31,38x. The amino acid taurine is an important intracellular osmoequivalent and the efflux of 3 H-taurine follows closely the time course of astrocytic volume reduction w31x. Astrocytes lack the ability to synthesize taurine but accumulate taurine efficiently w16x and taurine is released in vivo into the extracellular fluid in response to brain ischemia, seizures as well as after systemic water administration w1,19,20x.
0169-328Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 2 4 0 - X
M. Ding et al.r Molecular Brain Research 62 (1998) 77–81
78
The mechanisms which trigger RVD and the sensors for cell volume are largely unknown. Stretch-activated channels in the plasma membrane w35,36x, fusion of intracellular, transporter-containing vesicles with the plasma membrane w8x and the actin cytoskeleton have been proposed as effector mechanisms w3,22,26x. Microfilament disrupting agents, such as cytochalasin B, induce morphological changes in astrocytes w5,6x and affect the efflux of osmoequivalents in isoosmotic conditions w31x. The IFs change their bundling characteristics in swollen astrocytes w39x and the normally distinct network of GFAP immunoreactivity becomes unevenly diffuse w27x. We address here the role of IFs on the RVD of primary cultures of astrocytes from mice lacking GFAP andror vimentin w7,9,33x by monitoring the change in efflux of preloaded 3 H-taurine during exposure to a hypoosmotic medium. The RVD was significantly affected in astrocytes from GFAP–r–vim–r– mice, but not in astrocytes from mice lacking only GFAP or only vimentin.
2. Materials and methods 2.1. Preparation of astrocyte-enriched cultures from whole brain Wild type, GFAP–r– w33x, vimentin–r– w7x, or GFAP–r–vim–r– w9x mice were used, all hybrids between the C57BLr6 and 129 Ola strains. They were generated within the same litters to minimize genetic variation among the groups. Mice of postnatal day 1–2 were killed by decapitation and primary astrocyte-enriched cultures were prepared and maintained as described w32x. Total protein of the cultures was determined according to Lowry et al. w23x.
2.2. Efflux of 3H-taurine The efflux of 3 H-taurine was studied in three series of experiments, i.e., in 18 and 23 days primary cultures and in cultures 7 days after the first passage ŽTable 1.. The experiments were performed at 378C ŽMir 152 incubator, Sanyo, Japan.. The cultures were first washed three times with HEPES medium of the following composition ŽmM.: NaCl 122, KCl 3.3, CaCl 2 1.2, MgSO4 0.4, KH 2 PO4 1.2, HEPES 25 and glucose 10, pH 7.4. They were then loaded with 50 mM taurine Ž2 mCirml 3 H-taurine, sp. act. 21.9 Cirmmol, Dupont, NEN. for 180 min in the same medium after which they were washed three times. The perfusion system contained six cylindrical chambers Ždiameter 30 mm, height 1.2 mm, volume 0.85 ml., each having inlet and outlet for the perfusion medium, in the roof of the chamber, 25 mm apart. The chambers were perfused at 0.8 mlrmin with a multichannel peristaltic pump ŽMiniplus 2, Gilson Medical Electronics, France.. After 30 min perfusion with the original HEPES medium, a medium with reduced NaCl concentration Žminus 25 mM. was perfused for 12 min. Two-minute samples were collected. The cultures were then solubilized in 0.75 ml 1 N NaOH for radioactivity and protein determinations. The radioactivity was measured with liquid scintillation Ž1215 Rackbeta, LKB-Wallac, Finland.. The efflux of 3 H-taurine is expressed as radioactivity in each fraction in percent of the radioactivity present in the culture at the same time w29x. The increased efflux with the hypoosmotic medium is given in percent of that recorded during 6 min preceding the media change. 2.3. Determination of endogenous taurine The concentration of endogenous taurine was determined after separation of o-phtaldialdehyde derivated
Table 1 Characteristics of the cultures Series
Genotype
Number of mice Žnumber of. cultures per mouse.
Type of culture
Plating substrate
Days in culture
Total protein Žmgrculture. a
Total intracellular 3 H-taurine prior to perfusiona Ždpmrmg protein =10 3 .
3 H-taurine release during isotonic phase of perfusiona Ž%; fractional release.
a
wild type GFAP–r– GFAP–r–vim–r–
3 Ž2. 4 Ž2. 5 Ž2.
primary, unpassaged
glass
18
91.0 " 16.1 153.8 " 12.6 101.0 " 13.5
7.0 " 0.4 5.9 " 0.7 7.5 " 0.4
0.58 " 0.04 0.44 " 0.05 0.54 " 0.08
b
wild type GFAP–r– vimentin–r– GFAP–r–vim–r–
5 Ž2. 2 Ž2. 3 Ž2. 3 Ž2.
primary, unpassaged
glass
23
192.1 " 33.4 151.8 " 5.0 221 " 23.2 209.0 " 27.5
6.2 " 0.5 7.3 " 0.6 6.2 " 0.5 6.5 " 1.1
0.51 " 0.06 0.49 " 0.17 0.42 " 0.05 0.43 " 0.10
c
wild type GFAP–r– vimentin–r– GFAP–r–vim–r–
1 Ž4. 1 Ž4. 1 Ž4. 1 Ž4.
passaged once
plastic
31 q 7
129.4 " 2.9 115.2 " 8.2 115.3 " 7.1 139.1 " 13.3
6.1 " 0.5 7.9 " 0.5 5.8 " 0.2 9.1 " 0.6
0.88 " 0.10 1.02 " 0.09 0.78 " 0.05 0.69 " 0.13
a
S.E.M. refers to variation among individual mice Ža, b., or to variation among parallel cultures prepared from the same mouse Žc..
M. Ding et al.r Molecular Brain Research 62 (1998) 77–81
79
amino acids with liquid chromatography on a Nucleosil 5 C-18 column using gradient elution with increasing methanol Ž0–90%. in a Na-phosphate buffer Ž50 mM, pH 5.40.. A Varian ŽPalo Alto, CA, USA. LC 5000 chromatograph, a Wisp 810B autoinjector ŽWaters. and a Schoeffel FS 970 fluorometer were used. Quantification employed standards and Maxima 820 software ŽWaters.. 2.4. Statistical eÕaluation The results are presented as mean " S.E.M. The number of experiments are shown in Table 1. The groups were compared using ANOVA test for repeated measurement followed by Fisher PLSD. A difference was considered significant at P - 0.01.
3. Results 3
H-Taurine release was studied in 18 and 23-day-old primary astrocyte cultures Žseries a and b. and in cultures 7 days following the first passage Žseries c., in order to control for possible effects of protein concentration, cell age, density, etc. Total protein levels were similar in control and mutant cultures in all series ŽTable 1.. The accumulation of 3 H-taurine and the rate of efflux in isoosmotic medium did not differ significantly among the cultures ŽTable 1.. Exposure to hypoosmotic medium lead to a pronounced increase in the efflux of 3 H-taurine. In wild type cultures, the efflux increased by approximately 400%, 700% and 900% in series a, b and c, respectively and a similar increase was recorded with the GFAP–r– or vimentin–r– cultures ŽFig. 1.. However, the response was reduced by 27%, 46% and 25%, respectively, in the GFAP–r–vim– r– astrocyte cultures of the a–c series ŽFig. 1; P - 0.01.. The concentration of endogenous taurine was determined in the cultures since a concentration difference might affect the efflux of 3 H-taurine. The concentration was 63 " 4 and 75 " 4 nmolrmg protein in 10- and 20-day-old wild type cultures, respectively, and 71 " 5 and 71 " 4 nmolrmg protein in GFAP–r–vim–r– cultures of the corresponding age. Consequently, the levels of endogenous taurine in astrocytes appeared to be independent of the presence of GFAP and vimentin.
Fig. 1. 3 H-taurine release from primary cultures of astrocytes following hypotonic stress Žcorresponding to y25 mM NaCl.. a, b, c—independent experimental series Žsee Table 1.. Results are presented as mean"S.E.M. with the S.E.M. bars indicated in one direction only; the horizontal bar indicates the period of hypotonic stress. In all three experimental series, GFAP–r–vim–r– cultures exhibited lowered hypotonicity-induced 3 Htaurine release, decreased by 27%, 46% and 25% respectively, compared to wild type cultures Ž P - 0.01..
4. Discussion Primary and passaged cultures of astrocytes from mice deficient in GFAP andror vimentin were used to determine the impact of IF depletion on 3 H-taurine release following hypoosmotic stress. We have shown that the
80
M. Ding et al.r Molecular Brain Research 62 (1998) 77–81
efflux of 3 H-taurine was significantly lower in primary and passaged cultures from mice lacking both GFAP and vimentin. The accumulation of 3 H-taurine, the rate of its efflux in isoosmotic media and the content of endogenous taurine did not differ among the cultures. Similarly to reactive astrocytes in vivo, cultured astrocytes produce GFAP, vimentin and nestin which assemble into IFs. In cultured astrocytes from GFAP–r– mice, production of vimentin and nestin is unaffected and the two proteins assemble into IF network of normal appearance, but of reduced density w9,32x. IFs are also formed in astrocytes from vimentin–r– mice, however, in this case, composed solely of GFAP w9x. While astrocytes in culture both from GFAP–r– mice and from vimentin–r– mice form IFs, astrocytes from GFAP–r–vim–r– mice do not contain any IFs; vimentin appears to be the obligatory polymerization partner of nestin in astrocytes and consequently nestin cannot form IFs on its own w9x. The present data indicate that, decreased amount of IFs or differences in their composition Ž GFAP–Õimentin–nestin in wild type mice, Õimentin–nestin in GFAP–r– mice, or GFAP only in vimentin–r– mice. do not significantly influence 3 Htaurine release. In contrast, a total absence of IFs Žin GFAP–r–vim–r– mice. results in a substantial reduction of the RVD-related 3 H-taurine release in astrocytes. Mechanisms controlling the taurine efflux in astrocytes are not well understood. Loss of taurine following a hypoosmotic challenge seems to be passive, probably through a membrane channel which may be stretch-activated w36x. IFs run as rope-like structures within the cells and are connected to the plasma membrane, even though knowledge about the nature of such connections, or about IF-associated proteins in astrocytes, is at present very limited. It is possible that IFs participate in the opening of such membrane channels and this process would then be impaired in IF-free GFAP–r–vim–r– astrocytes. Alternatively, the absence of IFs may alter general mechanical properties of astrocytes and indirectly impair the opening of membrane channels for taurine. The importance of RVD in astrocytes in the context of pathological conditions accompanied by this phenomenon remains to be evaluated in vivo. GFAP–r–vim–r– mice, the astrocytes of which exhibit a deficit in the RVD function, at least in vitro, have a potential as a model to explore the role of glia in brain ischemia and other conditions in which brain edema is severe complication.
Acknowledgements We wish to thank Drs. Charles Babinet and Alain Privat for valuable discussions and Dr. Babinet for providing the vimentin–r– mice. Ms. Anita Palm is gratefully acknowledged for analyzing endogenous taurine levels. This study was supported by grants from the Swedish Medical Re-
search Council, projects No. 00164 Žto A.H.. and No. 11548 Žto M.P.., the Swedish Cancer Foundation, the Swedish Society for Medicine and Gustaf V’s Foundation. M.P. was supported by a fellowship from the Swedish Society for Medical Research.
References w1x P. Andine, M. Sandberg, R. Bagenholm, A. Lehmann, H. Hagberg, Intra- and extracellular changes of amino acids in the cerebral cortex of the neonatal rat during hypoxic-ischemia, Brain Res. Dev. Brain Res. 64 Ž1991. 115–120. w2x K. Ballanyi, P. Grafe, Cell volume regulation in the nervous system, Ren. Physiol. Biochem. 11 Ž1988. 142–157. w3x H.F. Cantiello, Role of actin filament organization in cell volume and ion channel regulation, J. Exp. Zool. 279 Ž1997. 425–435. w4x M.E. Chamberlin, K. Strange, Anisosmotic cell volume regulation: a comparative view, Am. J. Physiol. 257 Ž1989. C159–C173. w5x J. Ciesielski-Treska, M.F. Bader, D. Aunis, Microtubular organization in flat epitheloid and stellate process-bearing astrocytes in culture, Neurochem. Res. 7 Ž1982. 275–286. w6x J. Ciesielski-Treska, B. Guerold, D. Aunis, Immunofluorescence study on the organization of actin in astroglial cells in primary cultures, Neuroscience 7 Ž1982. 509–522. w7x E. Colucci-Guyon, M.M. Portier, I. Dunia, D. Paulin, S. Pournin, C. Babinet, Mice lacking vimentin develop and reproduce without an obvious phenotype, Cell 79 Ž1994. 679–694. w8x R.P. Czekay, E. Kinne-Saffran, R.K. Kinne, Membrane traffic and sorbitol release during osmo- and volume regulation in isolated rat renal inner medullary collecting duct cells, Eur. J. Cell Biol. 63 Ž1994. 20–31. w9x C. Eliasson, C. Sahlgren, C.-H. Berthold, C. Betsholtz, J. Eriksson, M. Pekny, Intermediate filament protein partnership in astrocytes, submitted. w10x L.F. Eng, R.S. Ghirnikar, GFAP and astrogliosis, Brain Pathol. 4 Ž1994. 229–237. w11x E. Fuchs, Keratins and the skin, Annu. Rev. Cell Dev. Biol. 11 Ž1995. 123–153. w12x E. Fuchs, D.W. Cleveland, A structural scaffolding of intermediate filaments in health and disease, Science 279 Ž1998. 514–519. w13x R. Ganz, M. Swain, P.G. Traber, M. Dal Canto, R.F. Butterworth, A. Blei, Ammonia-induced swelling of rat cerebral cortical slices: implications for the pathogenesis of brain edema in acute hepatic failure, Metab. Brain Dis. 4 Ž1989. 213–223. w14x M.E. Hatten, R.K. Liem, M.L. Shelanski, C.A. Mason, Astroglia in CNS injury, Glia 4 Ž1991. 233–243. w15x E. Hoffman, Volume Regulation in Cultured Cells, Academic Press, New York, 1991, pp. 124–180. w16x R.J. Huxtable, Taurine in the central nervous system and the mammalian actions of taurine, Prog. Neurobiol. 32 Ž1989. 471–533. w17x H.K. Kimelberg, Swelling and Volume Control in Brain Astroglial Cells, Springer, New York, 1991, pp. 81–117. w18x H.K. Kimelberg, B.R. Ransom, Physiological and Pathological Aspects of Astrocytic Swelling, Academic Press, Orlando, FL, 1986, pp. 129–166. w19x A. Lehmann, H. Isacsson, A. Hamberger, Effects of in vivo administration of kainic acid on the extracellular amino acid pool in the rabbit hippocampus, J. Neurochem. 40 Ž1983. 1314–1320. w20x A. Lehmann, C. Carlstrom, ¨ E.A. Nagelhus, O.P. Ottersen, Elevation of taurine in hippocampal extracellular fluid and cerebrospinal fluid of acutely hypoosmotic rats: contribution by influx from blood?, J. Neurochem. 56 Ž1991. 690–697. w21x W. Liedtke, W. Edelmann, P.L. Bieri, F.C. Chiu, N.J. Cowan, R. Kucherlapati, C.S. Raine, GFAP is necessary for the integrity of
M. Ding et al.r Molecular Brain Research 62 (1998) 77–81
w22x
w23x
w24x
w25x
w26x
w27x
w28x
w29x
w30x
w31x
CNS white matter architecture and long-term maintenance of myelination, Neuron 17 Ž1996. 607–615. M.A. Linshaw, C.A. Fogel, G.P. Downey, E.W. Koo, A.I. Gotlieb, Role of cytoskeleton in volume regulation of rabbit proximal tubule in dilute medium, Am. J. Physiol. 262 Ž1992. F144–F150. O.H. Lowry, N.J. Rosebrough, A.L. Farr, N.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–275. J.R. Marszalek, T.L. Williamson, M.K. Lee, Z. Xu, P.N. Hoffman, M.W. Becher, T.O. Crawford, D.W. Cleveland, Neurofilament subunit NF-H modulates axonal diameter by selectively slowing neurofilament transport, J. Cell Biol. 135 Ž1996. 711–724. M.A. McCall, R.G. Gregg, R.R. Behringer, M. Brenner, C.L. Delaney, E.J. Galbreath, C.L. Zhang, R.A. Pearce, S.Y. Chiu, A. Messing, Targeted deletion in astrocyte intermediate filament ŽGfap. alters neuronal physiology, Proc. Natl. Acad. Sci. USA 93 Ž1996. 6361–6366. J. Moran, M. Sabanero, I. Meza, H. Pasantes-Morales, Changes of actin cytoskeleton during swelling and regulatory volume decrease in cultured astrocytes, Am. J. Physiol. 271 Ž1996. C1901–C1907. L.J. Noble, J.J. Hall, S. Chen, P.H. Chan, Morphologic changes in cultured astrocytes after exposure to glutamate, J. Neurotrauma 9 Ž1992. 255–267. W.T. Norton, D.A. Aquino, I. Hozumi, F.C. Chiu, C.F. Brosnan, Quantitative aspects of reactive gliosis: a review, Neurochem. Res. 17 Ž1992. 877–885. E.R. O’Connor, H.K. Kimelberg, Role of calcium in astrocyte volume regulation and in the release of ions and amino acids, J. Neurosci. 13 Ž1993. 2638–2650. J.E. Olson, R. Sankar, D. Holtzman, A. James, D. Fleischhacker, Energy-dependent volume regulation in primary cultured cerebral astrocytes, J. Cell Physiol. 128 Ž1986. 209–215. H. Pasantes-Morales, J. Moran, A. Schousboe, Volume-sensitive
w32x
w33x
w34x
w35x w36x
w37x
w38x
w39x
81
release of taurine from cultured astrocytes: properties and mechanism, Glia 3 Ž1990. 427–432. M. Pekny, C. Eliasson, C.L. Chien, L.G. Kindblom, R. Liem, A. Hamberger, C. Betsholtz, GFAP-deficient astrocytes are capable of stellation in vitro when cocultured with neurons and exhibit a reduced amount of intermediate filaments and an increased cell saturation density, Exp. Cell Res. 239 Ž1998. 332–343. M. Pekny, P. Leveen, M. Pekna, C. Eliasson, C.H. Berthold, B. Westermark, C. Betsholtz, Mice lacking glial fibrillary acidic protein display astrocytes devoid of intermediate filaments but develop and reproduce normally, EMBO J. 14 Ž1995. 1590–1598. M. Pekny, K.A. Stanness, C. Eliasson, C. Betsholtz, D. Janigro, Impaired induction of blood–brain barrier properties in aortic endothelial cells by astrocytes from GFAP-deficient mice, Glia 22 Ž1998. 390–400. F. Sachs, Baroreceptor mechanisms at the cellular level, Fed. Proc. 46 Ž1987. 12–16. R. Sanchez-Olea, J. Moran, A. Schousboe, H. Pasantes-Morales, Hypoosmolarity-activated fluxes of taurine in astrocytes are mediated by diffusion, Neurosci. Lett. 130 Ž1991. 233–236. K. Shibuki, H. Gomi, L. Chen, S. Bao, J.J. Kim, H. Wakatsuki, T. Fujisaki, K. Fujimoto, A. Katoh, T. Ikeda, C. Chen, R.F. Thompson, S. Itohara, Deficient cerebellar long-term depression, impaired eyeblink conditioning, and normal motor coordination in GFAP mutant mice, Neuron 16 Ž1996. 587–599. D. Vitarella, D.J. DiRisio, H.K. Kimelberg, M. Aschner, Potassium and taurine release are highly correlated with regulatory volume decrease in neonatal primary rat astrocyte cultures, J. Neurochem. 63 Ž1994. 1143–1149. C.L. Willard-Mack, R.C. Koehler, T. Hirata, L.C. Cork, H. Takahashi, R.J. Traystman, S.W. Brusilow, Inhibition of glutamine synthetase reduces ammonia-induced astrocyte swelling in rat, Neuroscience 71 Ž1996. 589–599.
Research report
Altered taurine release following hypotonic stress in astrocytes from mice deficient for GFAP and vimentin Mei Ding
a,1
, Camilla Eliasson a
b,1
, Christer Betsholtz b, Anders Hamberger a , Milos Pekny
b,)
Department of Anatomy and Cell Biology, UniÕersity of Goteborg, PO Box 420, SE-405 30 Goteborg, Sweden ¨ ¨ b Department of Medical Biochemistry, UniÕersity of Goteborg, PO Box 440, SE-405 30 Goteborg, Sweden ¨ ¨ Accepted 1 September 1998
Abstract Astrocytes maintain their volume in response to changes in osmotic pressure in their environment by an affluxrinflux of ions and organic osmoequivalents. The initial swelling of an astrocyte transferred to a hypoosmotic medium is thus reversed within minutes. The mechanisms which trigger this process as well as the sensors for cell volume are largely unknown, however, the cytoskeleton appears to be involved. We have addressed the role of one component of the cytoskeleton, the intermediate filaments, in the maintenance of astrocytic cell volume. Astrocytes from wild type mice were compared with cells from mice deficient for either glial fibrillary acidic protein ŽGFAP–r– . or vimentin Žvimentin–r– . and with astrocytes from mice deficient for both proteins ŽGFAP–r–vim–r– .. Whereas GFAP–r– and vimentin–r– cultured or reactive astrocytes retain intermediate filaments, the GFAP–r–vim–r– astrocytes are completely devoid of these structures. The rate of efflux of the preloaded osmoequivalent 3 H-taurine from primary and passaged cultures of astrocytes was monitored. A reduction of NaCl Ž25 mM. in the perfusion medium led to a 400–900% increase of 3 H-taurine afflux in astrocytes from wild type mice. The stimulated efflux was not significantly affected in astrocytes from GFAP–r– or vimentin–r– mice. However, the efflux from astrocytes from GFAP–r–vim–r– mice was 25–46% lower than the wild type levels. The results strengthen the role of the cytoskeleton in astrocyte volume regulation and suggest an involvement of intermediate filaments in the process. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Astrocyte; Transgenic mouse; GFAP; Vimentin; Taurine; RVD Žregulatory volume decrease.
1. Introduction Changes in volume and shape of astrocytes is a common feature of CNS pathology, i.e., after hypoxia, ischemia, trauma, sustained seizures or liver failure w13,18x. Such conditions also involve increased production of the intermediate filament ŽIF. proteins, glial fibrillary acidic protein ŽGFAP. and vimentin w10,14,28x. Chemical composition of IFs reflects developmental and cell-type specificity, however the exact functions of this part of the cytoskeleton remain largely unknown. The role of IFs in the maintenance of mechanical integrity of cells and tissues has been recently established, primarily based on studies of keratin deficiencies in humans and on studies on transgenic mice w11,12x. Neurofilaments, the IFs of nerve
) C orresponding author. Fax: q 46-31-416108; [email protected] 1 These authors contributed equally to this work.
E-m ail:
cells, were found to be crucial for the modulation of the calibre of axons w24x, while GFAP was proposed to play a role in the inductionrmaintenance of the blood–brain barrier w21,34x as well as in modulation of neuronal functions, such as long-term depression w37x and long-term potentiation w25x. Astrocytes in culture respond with rapid swelling even to moderately hypoosmotic media but return, in this environment, to their original volume in 10–15 min w2,4,15,16,17,30x. This process, known as regulatory volume decrease ŽRVD., is of physiological importance for many cell types and involves an efflux of intracellular osmoequivalents w31,38x. The amino acid taurine is an important intracellular osmoequivalent and the efflux of 3 H-taurine follows closely the time course of astrocytic volume reduction w31x. Astrocytes lack the ability to synthesize taurine but accumulate taurine efficiently w16x and taurine is released in vivo into the extracellular fluid in response to brain ischemia, seizures as well as after systemic water administration w1,19,20x.
0169-328Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 9 - 3 2 8 X Ž 9 8 . 0 0 2 4 0 - X
M. Ding et al.r Molecular Brain Research 62 (1998) 77–81
78
The mechanisms which trigger RVD and the sensors for cell volume are largely unknown. Stretch-activated channels in the plasma membrane w35,36x, fusion of intracellular, transporter-containing vesicles with the plasma membrane w8x and the actin cytoskeleton have been proposed as effector mechanisms w3,22,26x. Microfilament disrupting agents, such as cytochalasin B, induce morphological changes in astrocytes w5,6x and affect the efflux of osmoequivalents in isoosmotic conditions w31x. The IFs change their bundling characteristics in swollen astrocytes w39x and the normally distinct network of GFAP immunoreactivity becomes unevenly diffuse w27x. We address here the role of IFs on the RVD of primary cultures of astrocytes from mice lacking GFAP andror vimentin w7,9,33x by monitoring the change in efflux of preloaded 3 H-taurine during exposure to a hypoosmotic medium. The RVD was significantly affected in astrocytes from GFAP–r–vim–r– mice, but not in astrocytes from mice lacking only GFAP or only vimentin.
2. Materials and methods 2.1. Preparation of astrocyte-enriched cultures from whole brain Wild type, GFAP–r– w33x, vimentin–r– w7x, or GFAP–r–vim–r– w9x mice were used, all hybrids between the C57BLr6 and 129 Ola strains. They were generated within the same litters to minimize genetic variation among the groups. Mice of postnatal day 1–2 were killed by decapitation and primary astrocyte-enriched cultures were prepared and maintained as described w32x. Total protein of the cultures was determined according to Lowry et al. w23x.
2.2. Efflux of 3H-taurine The efflux of 3 H-taurine was studied in three series of experiments, i.e., in 18 and 23 days primary cultures and in cultures 7 days after the first passage ŽTable 1.. The experiments were performed at 378C ŽMir 152 incubator, Sanyo, Japan.. The cultures were first washed three times with HEPES medium of the following composition ŽmM.: NaCl 122, KCl 3.3, CaCl 2 1.2, MgSO4 0.4, KH 2 PO4 1.2, HEPES 25 and glucose 10, pH 7.4. They were then loaded with 50 mM taurine Ž2 mCirml 3 H-taurine, sp. act. 21.9 Cirmmol, Dupont, NEN. for 180 min in the same medium after which they were washed three times. The perfusion system contained six cylindrical chambers Ždiameter 30 mm, height 1.2 mm, volume 0.85 ml., each having inlet and outlet for the perfusion medium, in the roof of the chamber, 25 mm apart. The chambers were perfused at 0.8 mlrmin with a multichannel peristaltic pump ŽMiniplus 2, Gilson Medical Electronics, France.. After 30 min perfusion with the original HEPES medium, a medium with reduced NaCl concentration Žminus 25 mM. was perfused for 12 min. Two-minute samples were collected. The cultures were then solubilized in 0.75 ml 1 N NaOH for radioactivity and protein determinations. The radioactivity was measured with liquid scintillation Ž1215 Rackbeta, LKB-Wallac, Finland.. The efflux of 3 H-taurine is expressed as radioactivity in each fraction in percent of the radioactivity present in the culture at the same time w29x. The increased efflux with the hypoosmotic medium is given in percent of that recorded during 6 min preceding the media change. 2.3. Determination of endogenous taurine The concentration of endogenous taurine was determined after separation of o-phtaldialdehyde derivated
Table 1 Characteristics of the cultures Series
Genotype
Number of mice Žnumber of. cultures per mouse.
Type of culture
Plating substrate
Days in culture
Total protein Žmgrculture. a
Total intracellular 3 H-taurine prior to perfusiona Ždpmrmg protein =10 3 .
3 H-taurine release during isotonic phase of perfusiona Ž%; fractional release.
a
wild type GFAP–r– GFAP–r–vim–r–
3 Ž2. 4 Ž2. 5 Ž2.
primary, unpassaged
glass
18
91.0 " 16.1 153.8 " 12.6 101.0 " 13.5
7.0 " 0.4 5.9 " 0.7 7.5 " 0.4
0.58 " 0.04 0.44 " 0.05 0.54 " 0.08
b
wild type GFAP–r– vimentin–r– GFAP–r–vim–r–
5 Ž2. 2 Ž2. 3 Ž2. 3 Ž2.
primary, unpassaged
glass
23
192.1 " 33.4 151.8 " 5.0 221 " 23.2 209.0 " 27.5
6.2 " 0.5 7.3 " 0.6 6.2 " 0.5 6.5 " 1.1
0.51 " 0.06 0.49 " 0.17 0.42 " 0.05 0.43 " 0.10
c
wild type GFAP–r– vimentin–r– GFAP–r–vim–r–
1 Ž4. 1 Ž4. 1 Ž4. 1 Ž4.
passaged once
plastic
31 q 7
129.4 " 2.9 115.2 " 8.2 115.3 " 7.1 139.1 " 13.3
6.1 " 0.5 7.9 " 0.5 5.8 " 0.2 9.1 " 0.6
0.88 " 0.10 1.02 " 0.09 0.78 " 0.05 0.69 " 0.13
a
S.E.M. refers to variation among individual mice Ža, b., or to variation among parallel cultures prepared from the same mouse Žc..
M. Ding et al.r Molecular Brain Research 62 (1998) 77–81
79
amino acids with liquid chromatography on a Nucleosil 5 C-18 column using gradient elution with increasing methanol Ž0–90%. in a Na-phosphate buffer Ž50 mM, pH 5.40.. A Varian ŽPalo Alto, CA, USA. LC 5000 chromatograph, a Wisp 810B autoinjector ŽWaters. and a Schoeffel FS 970 fluorometer were used. Quantification employed standards and Maxima 820 software ŽWaters.. 2.4. Statistical eÕaluation The results are presented as mean " S.E.M. The number of experiments are shown in Table 1. The groups were compared using ANOVA test for repeated measurement followed by Fisher PLSD. A difference was considered significant at P - 0.01.
3. Results 3
H-Taurine release was studied in 18 and 23-day-old primary astrocyte cultures Žseries a and b. and in cultures 7 days following the first passage Žseries c., in order to control for possible effects of protein concentration, cell age, density, etc. Total protein levels were similar in control and mutant cultures in all series ŽTable 1.. The accumulation of 3 H-taurine and the rate of efflux in isoosmotic medium did not differ significantly among the cultures ŽTable 1.. Exposure to hypoosmotic medium lead to a pronounced increase in the efflux of 3 H-taurine. In wild type cultures, the efflux increased by approximately 400%, 700% and 900% in series a, b and c, respectively and a similar increase was recorded with the GFAP–r– or vimentin–r– cultures ŽFig. 1.. However, the response was reduced by 27%, 46% and 25%, respectively, in the GFAP–r–vim– r– astrocyte cultures of the a–c series ŽFig. 1; P - 0.01.. The concentration of endogenous taurine was determined in the cultures since a concentration difference might affect the efflux of 3 H-taurine. The concentration was 63 " 4 and 75 " 4 nmolrmg protein in 10- and 20-day-old wild type cultures, respectively, and 71 " 5 and 71 " 4 nmolrmg protein in GFAP–r–vim–r– cultures of the corresponding age. Consequently, the levels of endogenous taurine in astrocytes appeared to be independent of the presence of GFAP and vimentin.
Fig. 1. 3 H-taurine release from primary cultures of astrocytes following hypotonic stress Žcorresponding to y25 mM NaCl.. a, b, c—independent experimental series Žsee Table 1.. Results are presented as mean"S.E.M. with the S.E.M. bars indicated in one direction only; the horizontal bar indicates the period of hypotonic stress. In all three experimental series, GFAP–r–vim–r– cultures exhibited lowered hypotonicity-induced 3 Htaurine release, decreased by 27%, 46% and 25% respectively, compared to wild type cultures Ž P - 0.01..
4. Discussion Primary and passaged cultures of astrocytes from mice deficient in GFAP andror vimentin were used to determine the impact of IF depletion on 3 H-taurine release following hypoosmotic stress. We have shown that the
80
M. Ding et al.r Molecular Brain Research 62 (1998) 77–81
efflux of 3 H-taurine was significantly lower in primary and passaged cultures from mice lacking both GFAP and vimentin. The accumulation of 3 H-taurine, the rate of its efflux in isoosmotic media and the content of endogenous taurine did not differ among the cultures. Similarly to reactive astrocytes in vivo, cultured astrocytes produce GFAP, vimentin and nestin which assemble into IFs. In cultured astrocytes from GFAP–r– mice, production of vimentin and nestin is unaffected and the two proteins assemble into IF network of normal appearance, but of reduced density w9,32x. IFs are also formed in astrocytes from vimentin–r– mice, however, in this case, composed solely of GFAP w9x. While astrocytes in culture both from GFAP–r– mice and from vimentin–r– mice form IFs, astrocytes from GFAP–r–vim–r– mice do not contain any IFs; vimentin appears to be the obligatory polymerization partner of nestin in astrocytes and consequently nestin cannot form IFs on its own w9x. The present data indicate that, decreased amount of IFs or differences in their composition Ž GFAP–Õimentin–nestin in wild type mice, Õimentin–nestin in GFAP–r– mice, or GFAP only in vimentin–r– mice. do not significantly influence 3 Htaurine release. In contrast, a total absence of IFs Žin GFAP–r–vim–r– mice. results in a substantial reduction of the RVD-related 3 H-taurine release in astrocytes. Mechanisms controlling the taurine efflux in astrocytes are not well understood. Loss of taurine following a hypoosmotic challenge seems to be passive, probably through a membrane channel which may be stretch-activated w36x. IFs run as rope-like structures within the cells and are connected to the plasma membrane, even though knowledge about the nature of such connections, or about IF-associated proteins in astrocytes, is at present very limited. It is possible that IFs participate in the opening of such membrane channels and this process would then be impaired in IF-free GFAP–r–vim–r– astrocytes. Alternatively, the absence of IFs may alter general mechanical properties of astrocytes and indirectly impair the opening of membrane channels for taurine. The importance of RVD in astrocytes in the context of pathological conditions accompanied by this phenomenon remains to be evaluated in vivo. GFAP–r–vim–r– mice, the astrocytes of which exhibit a deficit in the RVD function, at least in vitro, have a potential as a model to explore the role of glia in brain ischemia and other conditions in which brain edema is severe complication.
Acknowledgements We wish to thank Drs. Charles Babinet and Alain Privat for valuable discussions and Dr. Babinet for providing the vimentin–r– mice. Ms. Anita Palm is gratefully acknowledged for analyzing endogenous taurine levels. This study was supported by grants from the Swedish Medical Re-
search Council, projects No. 00164 Žto A.H.. and No. 11548 Žto M.P.., the Swedish Cancer Foundation, the Swedish Society for Medicine and Gustaf V’s Foundation. M.P. was supported by a fellowship from the Swedish Society for Medical Research.
References w1x P. Andine, M. Sandberg, R. Bagenholm, A. Lehmann, H. Hagberg, Intra- and extracellular changes of amino acids in the cerebral cortex of the neonatal rat during hypoxic-ischemia, Brain Res. Dev. Brain Res. 64 Ž1991. 115–120. w2x K. Ballanyi, P. Grafe, Cell volume regulation in the nervous system, Ren. Physiol. Biochem. 11 Ž1988. 142–157. w3x H.F. Cantiello, Role of actin filament organization in cell volume and ion channel regulation, J. Exp. Zool. 279 Ž1997. 425–435. w4x M.E. Chamberlin, K. Strange, Anisosmotic cell volume regulation: a comparative view, Am. J. Physiol. 257 Ž1989. C159–C173. w5x J. Ciesielski-Treska, M.F. Bader, D. Aunis, Microtubular organization in flat epitheloid and stellate process-bearing astrocytes in culture, Neurochem. Res. 7 Ž1982. 275–286. w6x J. Ciesielski-Treska, B. Guerold, D. Aunis, Immunofluorescence study on the organization of actin in astroglial cells in primary cultures, Neuroscience 7 Ž1982. 509–522. w7x E. Colucci-Guyon, M.M. Portier, I. Dunia, D. Paulin, S. Pournin, C. Babinet, Mice lacking vimentin develop and reproduce without an obvious phenotype, Cell 79 Ž1994. 679–694. w8x R.P. Czekay, E. Kinne-Saffran, R.K. Kinne, Membrane traffic and sorbitol release during osmo- and volume regulation in isolated rat renal inner medullary collecting duct cells, Eur. J. Cell Biol. 63 Ž1994. 20–31. w9x C. Eliasson, C. Sahlgren, C.-H. Berthold, C. Betsholtz, J. Eriksson, M. Pekny, Intermediate filament protein partnership in astrocytes, submitted. w10x L.F. Eng, R.S. Ghirnikar, GFAP and astrogliosis, Brain Pathol. 4 Ž1994. 229–237. w11x E. Fuchs, Keratins and the skin, Annu. Rev. Cell Dev. Biol. 11 Ž1995. 123–153. w12x E. Fuchs, D.W. Cleveland, A structural scaffolding of intermediate filaments in health and disease, Science 279 Ž1998. 514–519. w13x R. Ganz, M. Swain, P.G. Traber, M. Dal Canto, R.F. Butterworth, A. Blei, Ammonia-induced swelling of rat cerebral cortical slices: implications for the pathogenesis of brain edema in acute hepatic failure, Metab. Brain Dis. 4 Ž1989. 213–223. w14x M.E. Hatten, R.K. Liem, M.L. Shelanski, C.A. Mason, Astroglia in CNS injury, Glia 4 Ž1991. 233–243. w15x E. Hoffman, Volume Regulation in Cultured Cells, Academic Press, New York, 1991, pp. 124–180. w16x R.J. Huxtable, Taurine in the central nervous system and the mammalian actions of taurine, Prog. Neurobiol. 32 Ž1989. 471–533. w17x H.K. Kimelberg, Swelling and Volume Control in Brain Astroglial Cells, Springer, New York, 1991, pp. 81–117. w18x H.K. Kimelberg, B.R. Ransom, Physiological and Pathological Aspects of Astrocytic Swelling, Academic Press, Orlando, FL, 1986, pp. 129–166. w19x A. Lehmann, H. Isacsson, A. Hamberger, Effects of in vivo administration of kainic acid on the extracellular amino acid pool in the rabbit hippocampus, J. Neurochem. 40 Ž1983. 1314–1320. w20x A. Lehmann, C. Carlstrom, ¨ E.A. Nagelhus, O.P. Ottersen, Elevation of taurine in hippocampal extracellular fluid and cerebrospinal fluid of acutely hypoosmotic rats: contribution by influx from blood?, J. Neurochem. 56 Ž1991. 690–697. w21x W. Liedtke, W. Edelmann, P.L. Bieri, F.C. Chiu, N.J. Cowan, R. Kucherlapati, C.S. Raine, GFAP is necessary for the integrity of
M. Ding et al.r Molecular Brain Research 62 (1998) 77–81
w22x
w23x
w24x
w25x
w26x
w27x
w28x
w29x
w30x
w31x
CNS white matter architecture and long-term maintenance of myelination, Neuron 17 Ž1996. 607–615. M.A. Linshaw, C.A. Fogel, G.P. Downey, E.W. Koo, A.I. Gotlieb, Role of cytoskeleton in volume regulation of rabbit proximal tubule in dilute medium, Am. J. Physiol. 262 Ž1992. F144–F150. O.H. Lowry, N.J. Rosebrough, A.L. Farr, N.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 Ž1951. 265–275. J.R. Marszalek, T.L. Williamson, M.K. Lee, Z. Xu, P.N. Hoffman, M.W. Becher, T.O. Crawford, D.W. Cleveland, Neurofilament subunit NF-H modulates axonal diameter by selectively slowing neurofilament transport, J. Cell Biol. 135 Ž1996. 711–724. M.A. McCall, R.G. Gregg, R.R. Behringer, M. Brenner, C.L. Delaney, E.J. Galbreath, C.L. Zhang, R.A. Pearce, S.Y. Chiu, A. Messing, Targeted deletion in astrocyte intermediate filament ŽGfap. alters neuronal physiology, Proc. Natl. Acad. Sci. USA 93 Ž1996. 6361–6366. J. Moran, M. Sabanero, I. Meza, H. Pasantes-Morales, Changes of actin cytoskeleton during swelling and regulatory volume decrease in cultured astrocytes, Am. J. Physiol. 271 Ž1996. C1901–C1907. L.J. Noble, J.J. Hall, S. Chen, P.H. Chan, Morphologic changes in cultured astrocytes after exposure to glutamate, J. Neurotrauma 9 Ž1992. 255–267. W.T. Norton, D.A. Aquino, I. Hozumi, F.C. Chiu, C.F. Brosnan, Quantitative aspects of reactive gliosis: a review, Neurochem. Res. 17 Ž1992. 877–885. E.R. O’Connor, H.K. Kimelberg, Role of calcium in astrocyte volume regulation and in the release of ions and amino acids, J. Neurosci. 13 Ž1993. 2638–2650. J.E. Olson, R. Sankar, D. Holtzman, A. James, D. Fleischhacker, Energy-dependent volume regulation in primary cultured cerebral astrocytes, J. Cell Physiol. 128 Ž1986. 209–215. H. Pasantes-Morales, J. Moran, A. Schousboe, Volume-sensitive
w32x
w33x
w34x
w35x w36x
w37x
w38x
w39x
81
release of taurine from cultured astrocytes: properties and mechanism, Glia 3 Ž1990. 427–432. M. Pekny, C. Eliasson, C.L. Chien, L.G. Kindblom, R. Liem, A. Hamberger, C. Betsholtz, GFAP-deficient astrocytes are capable of stellation in vitro when cocultured with neurons and exhibit a reduced amount of intermediate filaments and an increased cell saturation density, Exp. Cell Res. 239 Ž1998. 332–343. M. Pekny, P. Leveen, M. Pekna, C. Eliasson, C.H. Berthold, B. Westermark, C. Betsholtz, Mice lacking glial fibrillary acidic protein display astrocytes devoid of intermediate filaments but develop and reproduce normally, EMBO J. 14 Ž1995. 1590–1598. M. Pekny, K.A. Stanness, C. Eliasson, C. Betsholtz, D. Janigro, Impaired induction of blood–brain barrier properties in aortic endothelial cells by astrocytes from GFAP-deficient mice, Glia 22 Ž1998. 390–400. F. Sachs, Baroreceptor mechanisms at the cellular level, Fed. Proc. 46 Ž1987. 12–16. R. Sanchez-Olea, J. Moran, A. Schousboe, H. Pasantes-Morales, Hypoosmolarity-activated fluxes of taurine in astrocytes are mediated by diffusion, Neurosci. Lett. 130 Ž1991. 233–236. K. Shibuki, H. Gomi, L. Chen, S. Bao, J.J. Kim, H. Wakatsuki, T. Fujisaki, K. Fujimoto, A. Katoh, T. Ikeda, C. Chen, R.F. Thompson, S. Itohara, Deficient cerebellar long-term depression, impaired eyeblink conditioning, and normal motor coordination in GFAP mutant mice, Neuron 16 Ž1996. 587–599. D. Vitarella, D.J. DiRisio, H.K. Kimelberg, M. Aschner, Potassium and taurine release are highly correlated with regulatory volume decrease in neonatal primary rat astrocyte cultures, J. Neurochem. 63 Ž1994. 1143–1149. C.L. Willard-Mack, R.C. Koehler, T. Hirata, L.C. Cork, H. Takahashi, R.J. Traystman, S.W. Brusilow, Inhibition of glutamine synthetase reduces ammonia-induced astrocyte swelling in rat, Neuroscience 71 Ž1996. 589–599.