A.C.H. Yu, L. Hertz, M.D. Norenberg, E. Sykova and S.G. Waxman (Eds.) Progress in Brain Research, Vol. 94 0 1992 Elsevier Science Publishers B.V. All rights reserved.
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CHAPTER 20
Release of exogenous and endogenous neurotransmitter amino acids from cultured astrocytes G. Levi, V. Gallo and M. Patrizio Neurobiology Section, Laboratory of Pathophysiology, Istituto Superiore di Sanita, 00161 Rome, Italy
Introduction Cell culture studies published in the last 9 years have provided evidence for the existence, in the rodent CNS, of two subpopulations of astrocytes, characterized by distinct morphological, antigenic and functional features and belonging to two different glial cell lineages (see Raff, 1989, for review). The two cell types were originally named type-1 and type 2 astrocytes by Raff and collaborators (Raff et al., 1983a,b)and most of the following literature has adopted this nomenclature (even if not always in an appropriate way). Most functional studies on astroglial cells have been performed using cultures enriched in type-1 astrocytes. Although the distinction between type-1 and type-2 astrocytes can not provide more than a preliminary and incomplete classification for the numerous and surely heterogeneous astroglial cells that can be grown in culture after dissociation from the newborn rodent brain, the possibility of recognizing these two cell types in culture, as well as that of obtaining cultures highly enriched in each of the two cell populations makes a comparison of the respective functional properties possible. Schematically, it can be said that type-1 astrocytes have an epithelioid morphology (in serum containing cultures, without the addition of “differentiating” agents such as cyclic AMP analogs), while type-2 astrocytes have an elaborate stellate shape (Raff et al., 1983a,b; Levi et al., 1983, 1986; Wilkin et al.,
1983; Aloisi et al., 1988a). Type-1 and type-2 astrocytes differ also in several other aspects: (i) they are recognized by different antibodies binding to different surface markers (Ran-2 antibodies bind to type-1 astrocytes (Raff et al., 1983a), while antitetanus toxin antibodies and the monoclonal antibodies A2B5, LBl and at times 04 bind to type-2 astrocytes (Raff et al., 1983a,b; Johnstone et al., 1986; Levi et al., 1986, 1987; Behar et al., 1988; Aloisi et al., 1988a,b; Trotter and Schachner, 1989); (ii) they express different extracellular matrix components (laminin and fibronectin can be expressed by type- 1 astrocytes, whereas chondroitin sulfate is present in type-2 astrocytes (Gallo et al., 1987; Gallo and Bertolotto, 1990); (iii) type-2, but not type-1 astrocytes accumulate the neurotransmitter GABA through an avid high-affinity transport system (Levi et al., 1983, 1986; Wilkin et al., 1983; Johnstone et al., 1986) and synthesize GABA through the putrescin pathway (Barres et al., 1990a); (iv) type-2, but not type-1 astrocytes have a high density of ionotropic non-NMDA receptors (Gallo et al., 1989; Usowicz et al., 1989; Barres et al., 1990a; Wyllie et al., 1991) while 0-adrenergic receptors are mainly expressed by type-1 astrocytes (Burgess and McCarthy, 1985; Trimmer and McCarthy, 1986); (v) type-1 and type-2 astrocytes have different excitability properties, for example they differ in the expression of voltage-dependent ionic channels (Barres et al., 1988, 1990a); (vi) type-1 and type-2 astrocytes differ in their properties as im-
244
munocompetent cells (Aloisiet al., 1988a,b; and unpublished results); and (vii) type-1 astrocytes are highly proliferative cells (in serum containing media) while type-2 astrocytes have only a minimal proliferative activity (Raff et al., 1983b; Wilkin et al., 1983; Aloisi et al., 1988a). It must be added, however, that several of the above mentioned astroglial properties can change with time in culture and with culture conditions (see, for example, Aloisi et al., 1988a; Barreset al., 1989; Wyllieetal., 1991). In vivo, astrocytes are in close contact with neuronal cell bodies, with their dendritic and axonic processes and with synaptic regions. The perineuronal milieu is likely to undergo substantial changes in its ionic and neurotransmitter content in relation to neuronal activity, and astrocytes are believed to have an important role in the removal of several neuroactive substances and of excess potassium from the extracellular environment (Hertz, 1982; Kimelberg and Norenberg, 1989). However, the recent finding that astrocytes express a variety of neurotransmitter receptors (for reviews, see Hertz et al., 1984; Murphy and Pearce, 1987; Barreset al., 1990b;Kettenmannet al., 1990; Wilkin et al., 1990) and that several neurotransmitters (Bowman and Kimelberg, 1984; Kettenmann and Schachner, 1985; Hosli et al., 1987, 1990; Kettenmann et al., 1990) as well as potassium (Martin et al., 1990)can depolarize their membranes, raises the possibility that astrocytes can actively respond to various environmental stimuli. It can be hypothesized that the functional response of astrocytes to substances present in the extracellular environment as a consequence of neuronal activity may in turn exert a modulating action on neuronal activity itself. One way by which this could occur is through the release by astrocyte of neuroactive or neuromodulatory substances. As an initial approach to test this hypothesis, we determined whether the activation of the excitatory amino acid receptor subtypes present on astrocytes (the non-NMDA receptors) induces the release of neuroactive amino acids from these cells. Our experiments were performed either on mixed astrocyte cultures obtained from the 8-day-old post-natal rat
cerebellum as described by Wilkin et al. (1983) or on purified subcultures of type-1 or type-2 astrocytes obtained from 1-day-oldpost-natal rat cerebral cortex as described by Aloisi et al. (1988a) and Agresti et al. (1991). Release of [3H]GABA induced by non-NMDA receptor agonists
In a first series of experiments we determined whether non-NMDA receptor agonists can stimulate the release of preloaded [3H]GABA from the two astrocyte types. Previous studies had shown that [3H]GABAis avidly accumulated by type-2 but not by type-1 astrocytes (Levi et al., 1983; Wilkin et al., 1983; Johnstoneet al., 1986). Exposureof 5-day cerebellar cultures comprising both type-1 and type2 astrocytes to micromolar concentrations of kainate (KA), quisqualate (QA) or amino-3hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) caused a dose-dependent and Na + -dependent release of [3H]GABA (Gallo et al., 1989, 1991). Exposure to KA of 8 - 10-day cultures, comprising almost exclusively type-1 astrocytes, did not evoke [3H]GABA release (Gallo et al., 1989). The lack of release in these cultures does not seem to be related to the scarce [3H]GABA accumulation by type-1 astrocytes, since also [3H]~-aspartate,which was avidly accumulated by both type-1 and type-2 astrocytes (Levietal., 1983;Wilkinet al., 1983),was released only by cultures comprising both types of astrocytes and not by cultures lacking type-2 astrocytes (Gallo et al., 1989). These observations were confirmed in purified cultures of cortical type1 and type-2 astrocytes (V. Gallo, M. Patrizio and G. Levi, unpublished results.) The effect of KA was selectively, although not totally, antagonized by kynurenic acid, while 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX) prevented the effects of all three agonists (Gallo et al., 1989, 1991). The above mentioned experiments indicated that exposure to non-NMDA receptor agonists induced release of preloaded [3H]GABA and [ 3 H ] ~ aspartate from type-2 astrocytes through a receptormediated mechanism. The reasons why type- 1
245
astrocytes did not respond to non-NMDA agonists may be either lack or scarcity of non-NMDA receptors, absence of coupling between receptor activation and amino acid release, or compartmentation of the radioactive amino acids in a non-releasable pool. The first hypothesis seems the most likely in view of the electrophysiological observations of some authors which showed a much higher density of ionotropic non-NMDA receptors in type-2 as compared to type-1 astrocytes (Usowiczet al., 1989; Barres et al., 1990a; Wyllie et al., 1991). It seemed important to determine the mechanism whereby [3H]GABA is released by glutamate agonists from type-2 astrocytes. The involvement of metabolotropic receptors in the QA-evoked release of [3H]GABAseems to be excluded by the fact that CNQX, which does not antagonize the metabolotropic effects of QA (Doble and Perrier, 1988; Palmer et al., 1988), prevented the releasing effect
of QA (Gallo et al., 1991). Moreover, metabolotropic QA receptors are known to be abundantly expressed by type-1 astrocytes (Pearce et al., 1986, 1990; Nicoletti et al., 1990), which did neither release [3H]GABA nor endogenous amino acids (see below) when exposed to QA. In a series of experiments we could also exclude the involvement of cyclic GMP in the action of KA. In fact, the level of the cyclic nucleotide was unaffected by KA in both type-1 and type-2 astrocytes, in spite of the fact that both type-1 and type-2 astrocytes contained guanylate cyclase, as shown both biochemically and by immunofluorescence (Gallo et al., 1991). These observations strongly suggest that the releasing effect of non-NMDA receptor agonists observed in type-2 astrocytes is related to their ionotropic action. In view of the absolute Na+-requirement of the glutamate agonist-evoked release of [3H]GABA, and of its scarce or absent Ca2+-dependence(Gallo
TABLE 1 Effect of kainic acid and quisqualic acid on the release of endogenous amino acids from type-2 astrocyte cultures Amino acid
ratio
release in the presence of agonist ( f antagonist) basal release
50 pM Kainate ASP GLU SER GLN GLY TAU ALA
*
1.57 0.17 (10) 1.97 0.23 (11) 1.45 f 0.12 (9) 1.10 0.10 (10) 1.63 f 0.22 (11) 1.65 f 0.22 (9) 1.58 0.14 (10)
* * +
50 pM Kainate 30 pM CNQX
1.12 f 0.18 (6)
1.15 f O.lO(6) 1.20 f 0.09 (4) 1.09 0.25 (4) 1.00 0.12 (4) 1.16 f 0.12 ( 5 ) 1.06 0.21 (6)
* * *
+
50 pM Quisqualate
1.73 2.18 1.36 1.44 1.89 1.57 1.60
* 0.19(12) f 0.18 (12) * 0.20(12) * 0.19 (11)
f 0.34 (12) f 0.18 (12) f 0.20(12)
50 pM Quisqualate 30 pM CNQX
+
1.23 * 0.17 (7) 1.15 * 0.12 (7) 1.17 1.06
+ 0.19(7)
* 0.11 (7)
1.20 *
1.09 f
0.97
*
0.13 (7)
0.24 (7) 0.01 (7)
Subcultures enriched in cortical type-2 astrocytes were exposed to kainate or quisqualate (in the presence or in the absence of CNQX) for 10 min. Amino acid concentrations were measured in the 5 min fraction preceding the aministration of drugs (basal release) and in a pool of the two 5 min fractions containing the drugs. The results are expressed as ratios between release in the presence of drugs and basal release. Means S.E.M.are presented. The number of culture dishes analyzed, derived from 2 to 6 different cell preparations, are given in parentheses. Statistical significance was evaluated by the paired Student’s t-test. In the case of kainate, the evoked release was statistically significant (see bold figures) for aspartate, glutamate, taurine and alanine ( P < 0.01), serine (P < 0.02) and glycine (P < 0.05). In the case of quisqualate, theevoked release was statistically significant (see bold figures) for aspartate, glutamate, taurine andalanine (P < 0.001), glutamine (P < 0.02) and glycine(P < 0.05). The release of asparagine, threonine, arginine, tyrosine, valine, phenylalanine, isoleucine and leucine was not significantly affected. In the presence of CNQX, no statistically significant evoked release was present (G. Levi and M. Patrizio, 1992).
*
246
et al., 1989),it seemed reasonable to test whether the release occurred through a carrier-mediated mechanism. In fact, GABA transport is known to be totally Na+-dependent (Kanner et al., 1983). This possibility was supported by two sets of experiments. One showed that exposure of the cultures preloaded with [3H]GABA to the GABA transport inhibitor nipecotic acid prevented the subsequent releasing action of KA. The inhibitory effect of nipecotic acid was progressively more pronounced as the exposure time to the GABA analog was increased, suggesting that nipecotic acid inhibited the GABA carrier at the internal side of the membrane (Gallo et al., 1991). The other set of experiments showed that replacement of Na+ by Li+ in the incubation medium prevented
the releasing action of KA and QA (Gallo et al., 1991). It is known that in the GABA transport system Na+ can not be replaced by Li+ (Pin and Bokaert, 1989), while glutamate-gated ion channels are similarly permeable to Na+ and Li+ (Mayer and Westbrook, 1987). In the Li+-containing medium, therefore, KA and QA are still capable of depolarizing type-2 astrocytes (Wyllie et al., 1991), but the absence of Na+ ions prevents the carriermediated release of [3H]GABA. In Na+-containing media the increased intracellular "a+] consequent to the increased Na+ influx through the receptorgated channels would instead facilitate the operation of the GABA carrier in an outward direction (Gallo et al., 1991).
TABLE I1 Lack of effect of kainic acid and quisqualic acid on the release of endogenous amino acids from type-1 astrocyte cultures Amino acid
ASP GLU ASN SER GLN GLY THR ARC TAU ALA TYR VAL PHE ILE LEU
Ratio
release in the presence of agonist ( f antagonist) basal release
50 pM Kainate
200 pM Kainate
50 pM Kainate dbcAMP-treated
50 pM Quisqualate
1.04 f 0.14 (9) 1.11 f 0.11 (9) 1.19 f 0.09 (10) 1.21 f 0.13 (10) 1.02 f 0.06 (10) 1.22 f 0.12 (9) 1.03 f 0.09 (10) 1.14 f 0.15 (6) 1.22 f 0.19 (8) 1.10 f 0.09 (8) 1.13 0.09 (7) 1.10 f 0.12 (7) 0.99 f 0.06 (7) 1.13 f 0.06 (8) 1.15 f 0.06 (8)
1.08 f 0.09 (4) 1.14 f 0.23 (5) 1.40 f 0.29 (4) 1.17 f 0.32 (4) 1.40 f 0.28 (5) 1.17 f 0.12 (5) 1.00 f 0.06 (5) 1.19 f 0.09 (4) 1.08 f 0.24 (4) 1.09 f 0.88 (4) 1.23 f 0.13 (5) 1.24 f O.lO(5) 1.06 f 0.07 (5) 1.14 f 0.08 (5) 1.22 f 0.12 (4)
0.73 f 0.09 (3) 0.94 f 0.08 (4) 0.93 f 0.04 (4) 0.90 f 0.09 (4) 0.80 f 0.05 (3) 1.01 f 0.10 (4) 0.86 f 0.08 (4) 1.06 f 0.18 (4) 1.56 f 0.49 (8) 1.02 f 0.16(4) 0.93 f 0.07 (4) 1.23 f 0.05 (3) 1.16 f 0.09 (3) 1.08 f 0.04 (3) 1.21 f 0.04 (3)
1.07 0.86 1.28 1.20 1.21 1.25 0.97 1.18 0.86 1.18 1.23 1.10 1.09 1.07 0.95
*
f 0.17 (8) f 0.06 (9) f 0.08 (7) f 0.13 (9) f 0.11 (7) f 0.11 (9) f 0.06 (9) f 0.12 (7) f 0.05 (9) f 0.11 (9)
f 0.12 (9) f 0.08 (6) f 0.07 (7) f 0.08 (6) f 0.06 (4)
Subcultures enriched in cortical type-1 astrocytes were exposed to kainate or quisqualate for 10 min. In one set of experiments the cultures had been pre-treated for 3 days with 1 mM dibutyryl cyclic AMP. Amino acid concentrations were measured in the 5 min fractions preceding the administration of drugs (basal release) and in a pool of the two 5 min fractions containing the drugs. The results are expressed as ratios between release in the presence of drugs and basal release. Means f S.E.M. are presented. The number of culture dishes analyzed, derived from 2 to 5 different cell preparations, is given in parentheses (G. Levi and M. Patrizio, 1992).
247
Release of endogenous amino acids induced by non-NMDA receptor agonists and by high [K+] Although some GABA synthesisthrough the putrescin pathway has been shown to occur in type-2 astrocytes from the rat optic nerve (Barres, 1990a), the astroglial concentration of GABA is very low, and GABA may not be the (or the only) endogenous amino acid whose release is induced by non-NMDA receptor activation, Moreover, the inability of KA and QA to release exogenous radioactive amino acids from cerebellar type-I astrocytes did not totally exclude the possibility that endogenous amino acids could be released from type-I astrocytes, particularly if these are prepared from another brain area. Therefore, parallel experiments were performed using secondary cultures highly enriched in cortical type-1 or type-2 astrocytes. The results of these experiments showed that exposure to 50 pM KA or QA caused a statistically significant, CNQX-
TABLE Ill Levels and basal release of endogenous amino acids from type-1 and type-2 astrocyte cultures Amino acid
Type-1 astrocyte levels (nmol/mg protein)
ASP GLU ASN SER GLN GLY THR TAU ALA
3.74 18.98 1.91 5.28 31.01 4.68 4.50 30.49 32.03
+ 0.83 (7)
i 1.19 (7) i 0.30 (7)
+ 0.33 (7) + 4.34 (7) f
1.08 (7)
i 0.40 (4)
* 1.69 (7)
+
1.17 (7)
Type-2 astrocyte levels (nmol/rng protein) 6.75 + 0.69 (7) 10.49 + 2.09 (6) 0.68 + 0.08 (6) 2.73 i 0.23 (6) 7.79 + 2.09 (6) 3.47 + 0.34 (7) 2.61 + 0.34 (6) 17.47 2.63 (7) 6.55 1.13 (7)
* *
Amino acid concentrations were measured in cell extracts by an HPLC procedure (levels). All the amino acids shown had statistically different levels in cell extracts of type-1 and type-2 astrocytes, as determined by the Student's t-test. The concentration of tyrosine, valine, phenylalanine, isoleucine and leucine was similar in the two cell types. Serine, glutamine and alanine (P < 0.001); aspartate, glutamate threonine and taurine (P < 0.01); glycine(P c 0.02). (From Levi andpatrizio, 1992.)
TABLE IV Effect of high [K'] and non-NMDA receptor agonists on cell volume of type-1 and type-2 astrocytes Condition
50 mM KCI 50 pM Kainate 50 pM Quisqualate
To Increase in cell volume
Type-1
Type-2
37 f 2 7 f 3
47 + 2 3 + 1
3*3
6 + 3
P
< 0.01 -
-
Subcultures highly enriched in type-1 or type-2 astrocytes were incubated for 10 min in the conditions listed. Cell volume was method. Means i measured by the ['4C]-3-O-methyl-glucose S.E.M. of 6 experiments run in duplicate are presented. Statistical significance of the difference in cell volume increase between type-1 and type-2 astrocytes was calculated by the Student's t-test (Levi and Patrizio, 1992).
sensitive increase in the release of a group of endogenous amino acids from type-2 (Table I), but not from type- 1 astrocytes (Table 11). Glutamateshowed the highest evoked release (about 100% increase over baseline), both in the case of KA and QA. Raising the concentration of KA up to 200 pM, or treating the type-1 astrocytes cultures with the differentiation agent dibutyryl cyclic AMP for 3 days did not change the release profile pattern (Table 11). In order to exclude the possibility that the lack of evoked release in type-1 astrocytes was related to much lower endogenous amino acid concentrations in these cells, compared to type-2 astrocytes, the levels of endogenous amino acids were measured in both types of culture. With the exception of aspartate, the concentration of several major endogenous amino acids was, however, subst'antially higher in type-1 than in type-2 astrocytes (Table 111). It has been reported that the release of taurine, glutamate and aspartate from type-1 astrocytes can be enhanced by high [K+] as a consequence of cell swelling (Pasantes-Morales and Schousboe, 1989; Martin et al., 1990). The possibility that the releasing activity of KA and QA observed in type-2 astrocytes was associated with cell swelling was ex-
248 TABLE V Effect of 50 mM KCL on the release of endogenous amino acids from type-1 and type-2 astrocytes Ratio
evoked release basal release
~~
Type-1 Taurine Glutamate Aspartate
1.9 1.7
1.3
+ 0.2 (Qb
* 0.2 (9)b
+ 0.1 (7)d
Type-2 2.3 + 0.3 (5)a 1.9 f 0.2 (7)b 1.3
+ 0.1 (5)‘
Subcultures enriched in type-1 or type-2 astrocytes were exposed to 50 mM KC1 (replacing an equimolar concentration of NaCI) for 10 min. Amino acid concentrations were measured in the 5 min fraction preceding depolarization (basal release) and in a pool of the two 5 min fractions containing high K + (evoked release). The results are expressed as ratios between evoked and S.E.M. are presented. The number of basal release. Means culture dishes analyzed (derived from 3 to 5 cell preparations) is given in parentheses. All the differences shown were statistically significant. Other amino acids were not affected (Levi and Patrizio, 1992). a P < 0.0001. P < 0.01. P < 0.02. P = 0.05.
*
cluded by the experiments reporteL in Table IV, which show that the two agonists did not alter cell volume in either type-1 or type-2 astrocytes in the experimental conditions adopted. On the other hand, exposure to 50 mM KCl (replacing an equimolar concentration of NaCl) induced cell swelling (Table IV) and release of taurine > glutamate > aspartate (Table V) in both type-1 and type-2 astrocytes, in agreement with the above mentioned findings of other authors. The fact that the increase in cell volume was higher in type-2 than in type-1 astrocytes may explain the higher stimulation of taurine and glutamate release observed in type-2 astrocytes. Interestingly, the endogenous level of taurine and glutamate was substantially lower in type-2 as compared to type-1 astrocytes (Table 111). If taurine and glutamate are involved in cell volume regulation in astrocytes, the lower endogenous con-
centration of these amino acids in type-2 astrocytes might account for the greater propensity of these cells to swell. It is worth noting that a number of neutral amino acids whose endogenous level was high (glutamine, glycine, threonine, alanine) were not released by high [K+ ]-induced swelling. This suggests that the charge of the amino acid may be important in this type of release. On the other hand, some of the above mentioned neutral amino acids were released by non-NMDA receptor agonists in type-2 astrocytes. The differences in the release profiles observed in the two conditions provide evidence for the specificity of the release patterns observed, and indicate that the mechanisms underlying the release process are different in the two depolarizing conditions (non-NMDA receptor activation and high [K +]-induced swelling, respectively). By analogy with the results obtained when studying the mechanism of KA-induced [3H]GABA release from type-2 astrocytes (Gallo et al., 1991) it can be suggested that the release of endogenous amino acids induced by non-NMDA receptor agonists occurs through the membrane carriers operating in an outward direction when the intracellular “a+] is increased following receptor activation. On the other hand, swelling-induced release is unlikely to be related to carrier-mediated transport processes (Kimelberg et al., 1990).
Conclusions In conclusion, our results indicate that reasonably low concentrations of non-NMDA receptor agonists stimulate the release of exogenous [3H]GABA and [3H]~-aspartate, and of a group of endogenous amino acids from a defined subpopulation of astrocytes (type-2 astrocytes), without causing cell swelling. Some of the amino acids released are directly neuroactive (glutamate, taurine), some may influence the activity of NMDA receptors (glycine, see Ascher and Johnson, 1990). It is premature to suggest what could be the functional implications of this observation in the living brain. Another relevant aspect of our study is that type-2
249
astrocytes respond to high [K+] by a volume increase and by releasing three neuroactive amino acids similarly to type-1 astrocytes. This is important in view of the fact that the two astroglial populations belong to different cell lineages, and show a number of functional differences, as outlined in the first part of the present report. Acknowledgements
This investigation was supported by grants of the Italian National Research Council no. 90.03191 .CT04 (Special project on “Neurotransmitter release mechanisms and control”) and no. 91.00469.PF40 (Project on “Aging”, subproject on “Gerontobiology”, ref. publ. 91-1-028). References Agresti, C., Aloisi, F. and Levi, G. (1991) Heterotypic and homotypic cellular interactions influencing the growth and differentiation of bipotential oligodendrocyte-type-2 astrocyte progenitors in culture. Dev. Biol., 144: 16 - 29. Aloisi, F., Agresti, C. and Levi, G. (1988a) Establishment, characterization, and evolution of cultures enriched in type-2 astrocytes. J. Neurosci. Res., 21: 188- 198. Aloisi, F., Agresti, C., D’Urso, D. and Levi, G. (1988b) Differentiation of bipotential glial precursors into oligodendrocytes is promoted by interaction with type-1 astrocytes in cerebellar cultures. Proc. Natl. Acad. Sci. U.S.A., 8 5 : 6167 - 6 171. Ascher, P. and Johnson, J . (1990) The structure of the NMDA receptor-channel complex. Prog. Cell Res., 1: 149- 158. Barres, B.A., Chun, L.L.Y. and Corey, D.P. (1988) Ion channel expression in white matter glia: 1. Type 2 astrocytes and oligodendrocytes. Glia, 1: 10 - 30. Barres, B.A., Chun, L.L.Y. and Corey, D.P. (1989) Calcium current in cortical astrocytes: induction by CAMP and neurotransmitters and permissive effect of serum. J. Neurosci., 9: 3169-3175. Barres, B.A., Koroshetz, W.J., Swartz, K. J., Chun, L.L.Y. and Corey, D.P. (1990a) Ion channel expression by white matter glia: the 0-2A glial progenitor cell. Neuron, 4: 507 - 524. Barres, B.A., Chun, L.L.Y. and Corey, D.P. (1990b) Ion channels in vertebrate glia. Annu. Res. Neurosci., 13: 441 -474. Behar, T., McMorris, F.A., Novothy, E.A., Barker, J.L. and Dubois-Dalcq, M. (1988) Growth and differentiation properties of 0-2A progenitors purified from rat cerebral hemispheres. J. Neurosci. Rex, 21: 168- 180. Bowman, C.L. and Kimelberg, H.K. (1984) Excitatory amino
acids directly depolarize rat brain astrocytes in primary culture. Nature, 311: 656-659. Burgess, S.K. and McCarthy, K.D. (1985) Autoradiographic quantitation of 0-adrenergic receptors on neural cells in primary cultures. I. Pharmacological studies of [’251]pindolol binding of individual astroglial cells. Brain Res., 335: 1-9. Doble, A. and Perrier, M.L. (1989) Pharmacology of excitatory amino acid receptors coupled to inositol phosphate metabolism in neonatal rat striatum. Neurochem. Int., 15: 1-8. Gallo, V. and Bertolotto, A . (1990) Extracellular matrix of cultured glial cells: selective expression of chondroitin 4sulfate by type-2 astrocytes and their progenitors. Exp. Cell Rex, 187: 211 -223. Gallo, V., Bertolotto, A. and Levi, G. (1987) The proteoglycan chondroitin sulfate is present in a subpopulation of cultured astrocytes and in their precursors. Dev. Biol., 123: 282 - 285. Gallo, V . , Giovannini, C., Suergiu, R. and Levi, G. (1989) Expression of excitatory amino acid receptors by cerebellar cells of the type-2 astrocyte cell lineage. J. Neurochem., 52: 1 - 9. Gallo V., Patrizio, M. and Levi, G. (1991) GABA release triggered by the activation of neuron-like non-NMDA receptors in cultured type 2 astrocytes is carrier mediated. Glia, 4: 245 - 255. Hertz, L. (1982) Astrocytes. In: A. Lajtha (Ed.), Handbook of Neurochemistry, Plenum, New York, pp. 319- 355. Hertz, L., Schousboe, I., Hertz, L. and Schousboe, A. (1984) Receptor expression in primary cultures of neurons or astrocytes. Prog. Neuropsychopharmacol. Biol. Psychiatry, 8: 521 -527. Hosli, L., Hosli, E., Baggi, M., Bassetti, C. and Uhr, M. (1987) Action of dopamine and serotonin on the membrane potential of cultured astrocytes. Exp. Brain Res., 65: 482-485. Hosli, L., Hosli, E., Redle, S., Rojas, J. and Schramek, H. (1990) Action of baclofen, GABA and antagonists on the membrane potential of cultured astrocytes of rat spinal cord. Neurosci. Lett., 117: 307 - 312. Johnstone, S.R., Levi, G., Wilkin, G.P., Schneider, A. and Ciotti, M.T. (1986) Subpopulations of rat cerebellar astrocytes in primary culture: morphology, cell surface antigens and 3H-GABA transport. Dev. Brain Res., 24: 63 - 75. Kanner, B.I., Bendahan, A. and Radian, R. (1983) Efflux and exchange of y-aminobutyric acid and nipecotic acid catalysed by synaptic plasma membrane vesicles isolated from immature rat brain. Biochim. Biophys. Acta, 731: 54- 62. Kettenmann, H. and Schachner, M. (1985) Pharmacological properties of y-aminobutyric acid-, glutamate-, and aspartateinduced depolarizations in cultured astrocytes. J . Neurosci., 5 : 3295 - 3301. Kettenmann, H., Backus, K.H., Berger, T.B., Sontheimer, H. and Schachner, M. (1990) Neurotransmitter receptors linked to ionic channels in cultured astrocytes: an electrophysiological approach. In: G. Levi (Ed.), Differentiation and Functions of GIial Cells, Wiley-Liss, New York, pp.
250 203 - 21 1. Kimelberg, K. and Norenberg, M.D. (1989) Astrocytes. Sci. Am., 260: 66-76. Kimelberg, H .K., Goderie, S.K., Higman, S., Pang, S. and Waniewski, R.A. (1990) Swelling-induced release of glutamate, aspartate, and taurine from astrocyte cultures. J . Neurosci., 10: 1583 - 1591. Levi, G. and Patrizio, M. (1992) Astrocyte heterogeneity: endogenous amino acid levels and release evoked by non-Nmethy1-D-aspartate receptor agonists and by potassiuminduced swelling in type-1 and type-2 astrocytes. J. Neurochem., 58: 1943 - 1952. Levi, G., Wilkin, G.P., Ciotti, M.T. and Johnstone, S. (1983) Enrichment of differentiated, stellate astrocytes in cerebellar interneuron cultures as studied by GFAP immunofluorescence and autoradiographic uptake patterns of ’H-~-aspartateand ’H-GABA. Dev. Brain Res., 10: 227 - 241. Levi, G., Gallo, V. and Ciotti, M.T. (1986) Bipotential precursors of putative fibrous astrocytes and oligodendrocytes in rat cerebellar cultures express distinct surface features and “neuron-like’’ y-aminobutyric acid transport. Proc. Natl. Acad. Sci. U.S.A., 83: 1504- 1508. Levi, G., Aloisi, F. and Wilkin, G.P. (1987) Differentiation of cerebellar bipotential glial precursors into oligodendrocytes in primary culture: developmental profile of surface antigens and mitotic activity. J. Neurosci. Res., 18: 407-417. Martin, D.L., Madelian, V., Seligmann, B. andShain, W. (1990) The role of osmotic pressure and membrane potential in K f stimulated taurine release from cultured astrocytes and LRM55 cells. J. Neurosci., 10: 571 -577. Mayer, M.L. and Westbrook, G.L. (1987)Permeation and block of N-methyl-D-aspartic acid receptor channels by divalent cation in mouse cultured central neurones. J. Physiol. (Lond.), 394: 501 - 527. Murphy, S. and Pearce, B. (1987) Functional receptors for neurotransmitters on astroglial cells. Neuroscience, 22: 381 - 394. Nicoletti, F., Magri, G., Ingrao, F., Bruno, V., Catania, M.V., Dell’Albani, P., Condorelli, D.F. and Avola, R. (1990) Excitatory amino acids stimulate inositol phospholipid hydrolysis and reduce proliferation in cultured astrocytes. J. Neurochem., 54: 771 -777. Palmer, E., Monaghan, D.T. and Cotman, C.W. (1988) Glutamate receptors and phosphoinositide metabolism: stimulation via quisqualate receptors is inhibited by N-methylD-aspartate receptor activation. Mol. BrainRes., 4: 161 - 166. Pasantes-Modes, H. and Schousboe, A. (1989) Release of
taurine from astrocytes during potassium-evoked swelling. Glia, 2: 45 - 50. Pearce, B., Albrecht, J., Morrow, C. and Murphy, S. (1986) Astrocyte glutamate receptor activation promotes inositol phospholipid turnover and calcium flux. Neurosci. L e f f . ,72: 335 - 340. Pearce, B., Morrow, C. and Murphy, S. (1990) Further characterization of excitatory amino acid receptors coupled to phosphoinositide metabolism in astrocytes. Neurosci. Lett., 113: 298-303. Pin, J.-P. and Bockaert, J. (1989) Two distinct mechanisms, differentially affected by excitatory amino acids, trigger GABA release from fetal mouse striatal neurons in primary culture. J. Neurosci., 9: 648 - 656. Raff, M.C. (1989) Glial cell diversification in the rat optic nerve. Science, 243: 1450- 1455. Raff, M.C., Abney, E.R., Cohen, J., Lindsay, R. and Noble, M. (1983a) Two types of astrocytes in cultures of developing rat white matters; differences in morphology, surface gangliosides, and growth characteristics. J. Neurosci., 3: 1289- 1300. Raff, M.C., Miller, R.H. and Noble, M. (1983b) A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium. Nature, 303: 390- 396. Trimmer, P.A. and McCarthy, K.D. (1986) , Immunocytochemically defined astroglia from fetal, newborn and young adult rats express 0-adrenergic receptors in vitro. Dev. Brain Res., 27: 151 - 165. Trotter, J. and Schachner, M. (1989) Cells positive for the 0 4 surface antigen isolated by cell sorting are able to differentiate into astrocytes or oligodendrocytes. Dev. Brain Res., 46: 115 - 122. Usowicz, M.M., Gallo, V. andcull-Candy, S.G.(1989) Multiple conductance channels in type-2 cerebellar astrocytes activated by excitatory amino acids. Nature, 339: 380- 383. Wilkin, G.P., Levi, G., Johnstone, S. and Riddle, P.N. (1983) Cerebellar astroglial cells in primary cultures: expression of different morphological appearances and differential ability to take up ’H-~-asparateand 3H-GABA. Dev. Brain Res., 10: 265 - 277. Wilkin, G.P., Marriott, D.R. and Cholewinski, A.J. (1990) Astrocyte heterogeneity. Trends Neurosci., 13: 43 - 45. Wyllie, D. J.A., Mathie, A., Symonds, C.J. and Cull-Candy, S.G. (1991) Activation of glutamate receptors and glutamate uptake in identified macroglial cells in rat cerebellar cultures. J. Physiol. (Lond.), 432: 235 - 258.
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CHAPTER 20
Release of exogenous and endogenous neurotransmitter amino acids from cultured astrocytes G. Levi, V. Gallo and M. Patrizio Neurobiology Section, Laboratory of Pathophysiology, Istituto Superiore di Sanita, 00161 Rome, Italy
Introduction Cell culture studies published in the last 9 years have provided evidence for the existence, in the rodent CNS, of two subpopulations of astrocytes, characterized by distinct morphological, antigenic and functional features and belonging to two different glial cell lineages (see Raff, 1989, for review). The two cell types were originally named type-1 and type 2 astrocytes by Raff and collaborators (Raff et al., 1983a,b)and most of the following literature has adopted this nomenclature (even if not always in an appropriate way). Most functional studies on astroglial cells have been performed using cultures enriched in type-1 astrocytes. Although the distinction between type-1 and type-2 astrocytes can not provide more than a preliminary and incomplete classification for the numerous and surely heterogeneous astroglial cells that can be grown in culture after dissociation from the newborn rodent brain, the possibility of recognizing these two cell types in culture, as well as that of obtaining cultures highly enriched in each of the two cell populations makes a comparison of the respective functional properties possible. Schematically, it can be said that type-1 astrocytes have an epithelioid morphology (in serum containing cultures, without the addition of “differentiating” agents such as cyclic AMP analogs), while type-2 astrocytes have an elaborate stellate shape (Raff et al., 1983a,b; Levi et al., 1983, 1986; Wilkin et al.,
1983; Aloisi et al., 1988a). Type-1 and type-2 astrocytes differ also in several other aspects: (i) they are recognized by different antibodies binding to different surface markers (Ran-2 antibodies bind to type-1 astrocytes (Raff et al., 1983a), while antitetanus toxin antibodies and the monoclonal antibodies A2B5, LBl and at times 04 bind to type-2 astrocytes (Raff et al., 1983a,b; Johnstone et al., 1986; Levi et al., 1986, 1987; Behar et al., 1988; Aloisi et al., 1988a,b; Trotter and Schachner, 1989); (ii) they express different extracellular matrix components (laminin and fibronectin can be expressed by type- 1 astrocytes, whereas chondroitin sulfate is present in type-2 astrocytes (Gallo et al., 1987; Gallo and Bertolotto, 1990); (iii) type-2, but not type-1 astrocytes accumulate the neurotransmitter GABA through an avid high-affinity transport system (Levi et al., 1983, 1986; Wilkin et al., 1983; Johnstone et al., 1986) and synthesize GABA through the putrescin pathway (Barres et al., 1990a); (iv) type-2, but not type-1 astrocytes have a high density of ionotropic non-NMDA receptors (Gallo et al., 1989; Usowicz et al., 1989; Barres et al., 1990a; Wyllie et al., 1991) while 0-adrenergic receptors are mainly expressed by type-1 astrocytes (Burgess and McCarthy, 1985; Trimmer and McCarthy, 1986); (v) type-1 and type-2 astrocytes have different excitability properties, for example they differ in the expression of voltage-dependent ionic channels (Barres et al., 1988, 1990a); (vi) type-1 and type-2 astrocytes differ in their properties as im-
244
munocompetent cells (Aloisiet al., 1988a,b; and unpublished results); and (vii) type-1 astrocytes are highly proliferative cells (in serum containing media) while type-2 astrocytes have only a minimal proliferative activity (Raff et al., 1983b; Wilkin et al., 1983; Aloisi et al., 1988a). It must be added, however, that several of the above mentioned astroglial properties can change with time in culture and with culture conditions (see, for example, Aloisi et al., 1988a; Barreset al., 1989; Wyllieetal., 1991). In vivo, astrocytes are in close contact with neuronal cell bodies, with their dendritic and axonic processes and with synaptic regions. The perineuronal milieu is likely to undergo substantial changes in its ionic and neurotransmitter content in relation to neuronal activity, and astrocytes are believed to have an important role in the removal of several neuroactive substances and of excess potassium from the extracellular environment (Hertz, 1982; Kimelberg and Norenberg, 1989). However, the recent finding that astrocytes express a variety of neurotransmitter receptors (for reviews, see Hertz et al., 1984; Murphy and Pearce, 1987; Barreset al., 1990b;Kettenmannet al., 1990; Wilkin et al., 1990) and that several neurotransmitters (Bowman and Kimelberg, 1984; Kettenmann and Schachner, 1985; Hosli et al., 1987, 1990; Kettenmann et al., 1990) as well as potassium (Martin et al., 1990)can depolarize their membranes, raises the possibility that astrocytes can actively respond to various environmental stimuli. It can be hypothesized that the functional response of astrocytes to substances present in the extracellular environment as a consequence of neuronal activity may in turn exert a modulating action on neuronal activity itself. One way by which this could occur is through the release by astrocyte of neuroactive or neuromodulatory substances. As an initial approach to test this hypothesis, we determined whether the activation of the excitatory amino acid receptor subtypes present on astrocytes (the non-NMDA receptors) induces the release of neuroactive amino acids from these cells. Our experiments were performed either on mixed astrocyte cultures obtained from the 8-day-old post-natal rat
cerebellum as described by Wilkin et al. (1983) or on purified subcultures of type-1 or type-2 astrocytes obtained from 1-day-oldpost-natal rat cerebral cortex as described by Aloisi et al. (1988a) and Agresti et al. (1991). Release of [3H]GABA induced by non-NMDA receptor agonists
In a first series of experiments we determined whether non-NMDA receptor agonists can stimulate the release of preloaded [3H]GABA from the two astrocyte types. Previous studies had shown that [3H]GABAis avidly accumulated by type-2 but not by type-1 astrocytes (Levi et al., 1983; Wilkin et al., 1983; Johnstoneet al., 1986). Exposureof 5-day cerebellar cultures comprising both type-1 and type2 astrocytes to micromolar concentrations of kainate (KA), quisqualate (QA) or amino-3hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) caused a dose-dependent and Na + -dependent release of [3H]GABA (Gallo et al., 1989, 1991). Exposure to KA of 8 - 10-day cultures, comprising almost exclusively type-1 astrocytes, did not evoke [3H]GABA release (Gallo et al., 1989). The lack of release in these cultures does not seem to be related to the scarce [3H]GABA accumulation by type-1 astrocytes, since also [3H]~-aspartate,which was avidly accumulated by both type-1 and type-2 astrocytes (Levietal., 1983;Wilkinet al., 1983),was released only by cultures comprising both types of astrocytes and not by cultures lacking type-2 astrocytes (Gallo et al., 1989). These observations were confirmed in purified cultures of cortical type1 and type-2 astrocytes (V. Gallo, M. Patrizio and G. Levi, unpublished results.) The effect of KA was selectively, although not totally, antagonized by kynurenic acid, while 6-cyano-2,3-dihydroxy-7-nitroquinoxaline (CNQX) prevented the effects of all three agonists (Gallo et al., 1989, 1991). The above mentioned experiments indicated that exposure to non-NMDA receptor agonists induced release of preloaded [3H]GABA and [ 3 H ] ~ aspartate from type-2 astrocytes through a receptormediated mechanism. The reasons why type- 1
245
astrocytes did not respond to non-NMDA agonists may be either lack or scarcity of non-NMDA receptors, absence of coupling between receptor activation and amino acid release, or compartmentation of the radioactive amino acids in a non-releasable pool. The first hypothesis seems the most likely in view of the electrophysiological observations of some authors which showed a much higher density of ionotropic non-NMDA receptors in type-2 as compared to type-1 astrocytes (Usowiczet al., 1989; Barres et al., 1990a; Wyllie et al., 1991). It seemed important to determine the mechanism whereby [3H]GABA is released by glutamate agonists from type-2 astrocytes. The involvement of metabolotropic receptors in the QA-evoked release of [3H]GABAseems to be excluded by the fact that CNQX, which does not antagonize the metabolotropic effects of QA (Doble and Perrier, 1988; Palmer et al., 1988), prevented the releasing effect
of QA (Gallo et al., 1991). Moreover, metabolotropic QA receptors are known to be abundantly expressed by type-1 astrocytes (Pearce et al., 1986, 1990; Nicoletti et al., 1990), which did neither release [3H]GABA nor endogenous amino acids (see below) when exposed to QA. In a series of experiments we could also exclude the involvement of cyclic GMP in the action of KA. In fact, the level of the cyclic nucleotide was unaffected by KA in both type-1 and type-2 astrocytes, in spite of the fact that both type-1 and type-2 astrocytes contained guanylate cyclase, as shown both biochemically and by immunofluorescence (Gallo et al., 1991). These observations strongly suggest that the releasing effect of non-NMDA receptor agonists observed in type-2 astrocytes is related to their ionotropic action. In view of the absolute Na+-requirement of the glutamate agonist-evoked release of [3H]GABA, and of its scarce or absent Ca2+-dependence(Gallo
TABLE 1 Effect of kainic acid and quisqualic acid on the release of endogenous amino acids from type-2 astrocyte cultures Amino acid
ratio
release in the presence of agonist ( f antagonist) basal release
50 pM Kainate ASP GLU SER GLN GLY TAU ALA
*
1.57 0.17 (10) 1.97 0.23 (11) 1.45 f 0.12 (9) 1.10 0.10 (10) 1.63 f 0.22 (11) 1.65 f 0.22 (9) 1.58 0.14 (10)
* * +
50 pM Kainate 30 pM CNQX
1.12 f 0.18 (6)
1.15 f O.lO(6) 1.20 f 0.09 (4) 1.09 0.25 (4) 1.00 0.12 (4) 1.16 f 0.12 ( 5 ) 1.06 0.21 (6)
* * *
+
50 pM Quisqualate
1.73 2.18 1.36 1.44 1.89 1.57 1.60
* 0.19(12) f 0.18 (12) * 0.20(12) * 0.19 (11)
f 0.34 (12) f 0.18 (12) f 0.20(12)
50 pM Quisqualate 30 pM CNQX
+
1.23 * 0.17 (7) 1.15 * 0.12 (7) 1.17 1.06
+ 0.19(7)
* 0.11 (7)
1.20 *
1.09 f
0.97
*
0.13 (7)
0.24 (7) 0.01 (7)
Subcultures enriched in cortical type-2 astrocytes were exposed to kainate or quisqualate (in the presence or in the absence of CNQX) for 10 min. Amino acid concentrations were measured in the 5 min fraction preceding the aministration of drugs (basal release) and in a pool of the two 5 min fractions containing the drugs. The results are expressed as ratios between release in the presence of drugs and basal release. Means S.E.M.are presented. The number of culture dishes analyzed, derived from 2 to 6 different cell preparations, are given in parentheses. Statistical significance was evaluated by the paired Student’s t-test. In the case of kainate, the evoked release was statistically significant (see bold figures) for aspartate, glutamate, taurine and alanine ( P < 0.01), serine (P < 0.02) and glycine (P < 0.05). In the case of quisqualate, theevoked release was statistically significant (see bold figures) for aspartate, glutamate, taurine andalanine (P < 0.001), glutamine (P < 0.02) and glycine(P < 0.05). The release of asparagine, threonine, arginine, tyrosine, valine, phenylalanine, isoleucine and leucine was not significantly affected. In the presence of CNQX, no statistically significant evoked release was present (G. Levi and M. Patrizio, 1992).
*
246
et al., 1989),it seemed reasonable to test whether the release occurred through a carrier-mediated mechanism. In fact, GABA transport is known to be totally Na+-dependent (Kanner et al., 1983). This possibility was supported by two sets of experiments. One showed that exposure of the cultures preloaded with [3H]GABA to the GABA transport inhibitor nipecotic acid prevented the subsequent releasing action of KA. The inhibitory effect of nipecotic acid was progressively more pronounced as the exposure time to the GABA analog was increased, suggesting that nipecotic acid inhibited the GABA carrier at the internal side of the membrane (Gallo et al., 1991). The other set of experiments showed that replacement of Na+ by Li+ in the incubation medium prevented
the releasing action of KA and QA (Gallo et al., 1991). It is known that in the GABA transport system Na+ can not be replaced by Li+ (Pin and Bokaert, 1989), while glutamate-gated ion channels are similarly permeable to Na+ and Li+ (Mayer and Westbrook, 1987). In the Li+-containing medium, therefore, KA and QA are still capable of depolarizing type-2 astrocytes (Wyllie et al., 1991), but the absence of Na+ ions prevents the carriermediated release of [3H]GABA. In Na+-containing media the increased intracellular "a+] consequent to the increased Na+ influx through the receptorgated channels would instead facilitate the operation of the GABA carrier in an outward direction (Gallo et al., 1991).
TABLE I1 Lack of effect of kainic acid and quisqualic acid on the release of endogenous amino acids from type-1 astrocyte cultures Amino acid
ASP GLU ASN SER GLN GLY THR ARC TAU ALA TYR VAL PHE ILE LEU
Ratio
release in the presence of agonist ( f antagonist) basal release
50 pM Kainate
200 pM Kainate
50 pM Kainate dbcAMP-treated
50 pM Quisqualate
1.04 f 0.14 (9) 1.11 f 0.11 (9) 1.19 f 0.09 (10) 1.21 f 0.13 (10) 1.02 f 0.06 (10) 1.22 f 0.12 (9) 1.03 f 0.09 (10) 1.14 f 0.15 (6) 1.22 f 0.19 (8) 1.10 f 0.09 (8) 1.13 0.09 (7) 1.10 f 0.12 (7) 0.99 f 0.06 (7) 1.13 f 0.06 (8) 1.15 f 0.06 (8)
1.08 f 0.09 (4) 1.14 f 0.23 (5) 1.40 f 0.29 (4) 1.17 f 0.32 (4) 1.40 f 0.28 (5) 1.17 f 0.12 (5) 1.00 f 0.06 (5) 1.19 f 0.09 (4) 1.08 f 0.24 (4) 1.09 f 0.88 (4) 1.23 f 0.13 (5) 1.24 f O.lO(5) 1.06 f 0.07 (5) 1.14 f 0.08 (5) 1.22 f 0.12 (4)
0.73 f 0.09 (3) 0.94 f 0.08 (4) 0.93 f 0.04 (4) 0.90 f 0.09 (4) 0.80 f 0.05 (3) 1.01 f 0.10 (4) 0.86 f 0.08 (4) 1.06 f 0.18 (4) 1.56 f 0.49 (8) 1.02 f 0.16(4) 0.93 f 0.07 (4) 1.23 f 0.05 (3) 1.16 f 0.09 (3) 1.08 f 0.04 (3) 1.21 f 0.04 (3)
1.07 0.86 1.28 1.20 1.21 1.25 0.97 1.18 0.86 1.18 1.23 1.10 1.09 1.07 0.95
*
f 0.17 (8) f 0.06 (9) f 0.08 (7) f 0.13 (9) f 0.11 (7) f 0.11 (9) f 0.06 (9) f 0.12 (7) f 0.05 (9) f 0.11 (9)
f 0.12 (9) f 0.08 (6) f 0.07 (7) f 0.08 (6) f 0.06 (4)
Subcultures enriched in cortical type-1 astrocytes were exposed to kainate or quisqualate for 10 min. In one set of experiments the cultures had been pre-treated for 3 days with 1 mM dibutyryl cyclic AMP. Amino acid concentrations were measured in the 5 min fractions preceding the administration of drugs (basal release) and in a pool of the two 5 min fractions containing the drugs. The results are expressed as ratios between release in the presence of drugs and basal release. Means f S.E.M. are presented. The number of culture dishes analyzed, derived from 2 to 5 different cell preparations, is given in parentheses (G. Levi and M. Patrizio, 1992).
247
Release of endogenous amino acids induced by non-NMDA receptor agonists and by high [K+] Although some GABA synthesisthrough the putrescin pathway has been shown to occur in type-2 astrocytes from the rat optic nerve (Barres, 1990a), the astroglial concentration of GABA is very low, and GABA may not be the (or the only) endogenous amino acid whose release is induced by non-NMDA receptor activation, Moreover, the inability of KA and QA to release exogenous radioactive amino acids from cerebellar type-I astrocytes did not totally exclude the possibility that endogenous amino acids could be released from type-I astrocytes, particularly if these are prepared from another brain area. Therefore, parallel experiments were performed using secondary cultures highly enriched in cortical type-1 or type-2 astrocytes. The results of these experiments showed that exposure to 50 pM KA or QA caused a statistically significant, CNQX-
TABLE Ill Levels and basal release of endogenous amino acids from type-1 and type-2 astrocyte cultures Amino acid
Type-1 astrocyte levels (nmol/mg protein)
ASP GLU ASN SER GLN GLY THR TAU ALA
3.74 18.98 1.91 5.28 31.01 4.68 4.50 30.49 32.03
+ 0.83 (7)
i 1.19 (7) i 0.30 (7)
+ 0.33 (7) + 4.34 (7) f
1.08 (7)
i 0.40 (4)
* 1.69 (7)
+
1.17 (7)
Type-2 astrocyte levels (nmol/rng protein) 6.75 + 0.69 (7) 10.49 + 2.09 (6) 0.68 + 0.08 (6) 2.73 i 0.23 (6) 7.79 + 2.09 (6) 3.47 + 0.34 (7) 2.61 + 0.34 (6) 17.47 2.63 (7) 6.55 1.13 (7)
* *
Amino acid concentrations were measured in cell extracts by an HPLC procedure (levels). All the amino acids shown had statistically different levels in cell extracts of type-1 and type-2 astrocytes, as determined by the Student's t-test. The concentration of tyrosine, valine, phenylalanine, isoleucine and leucine was similar in the two cell types. Serine, glutamine and alanine (P < 0.001); aspartate, glutamate threonine and taurine (P < 0.01); glycine(P c 0.02). (From Levi andpatrizio, 1992.)
TABLE IV Effect of high [K'] and non-NMDA receptor agonists on cell volume of type-1 and type-2 astrocytes Condition
50 mM KCI 50 pM Kainate 50 pM Quisqualate
To Increase in cell volume
Type-1
Type-2
37 f 2 7 f 3
47 + 2 3 + 1
3*3
6 + 3
P
< 0.01 -
-
Subcultures highly enriched in type-1 or type-2 astrocytes were incubated for 10 min in the conditions listed. Cell volume was method. Means i measured by the ['4C]-3-O-methyl-glucose S.E.M. of 6 experiments run in duplicate are presented. Statistical significance of the difference in cell volume increase between type-1 and type-2 astrocytes was calculated by the Student's t-test (Levi and Patrizio, 1992).
sensitive increase in the release of a group of endogenous amino acids from type-2 (Table I), but not from type- 1 astrocytes (Table 11). Glutamateshowed the highest evoked release (about 100% increase over baseline), both in the case of KA and QA. Raising the concentration of KA up to 200 pM, or treating the type-1 astrocytes cultures with the differentiation agent dibutyryl cyclic AMP for 3 days did not change the release profile pattern (Table 11). In order to exclude the possibility that the lack of evoked release in type-1 astrocytes was related to much lower endogenous amino acid concentrations in these cells, compared to type-2 astrocytes, the levels of endogenous amino acids were measured in both types of culture. With the exception of aspartate, the concentration of several major endogenous amino acids was, however, subst'antially higher in type-1 than in type-2 astrocytes (Table 111). It has been reported that the release of taurine, glutamate and aspartate from type-1 astrocytes can be enhanced by high [K+] as a consequence of cell swelling (Pasantes-Morales and Schousboe, 1989; Martin et al., 1990). The possibility that the releasing activity of KA and QA observed in type-2 astrocytes was associated with cell swelling was ex-
248 TABLE V Effect of 50 mM KCL on the release of endogenous amino acids from type-1 and type-2 astrocytes Ratio
evoked release basal release
~~
Type-1 Taurine Glutamate Aspartate
1.9 1.7
1.3
+ 0.2 (Qb
* 0.2 (9)b
+ 0.1 (7)d
Type-2 2.3 + 0.3 (5)a 1.9 f 0.2 (7)b 1.3
+ 0.1 (5)‘
Subcultures enriched in type-1 or type-2 astrocytes were exposed to 50 mM KC1 (replacing an equimolar concentration of NaCI) for 10 min. Amino acid concentrations were measured in the 5 min fraction preceding depolarization (basal release) and in a pool of the two 5 min fractions containing high K + (evoked release). The results are expressed as ratios between evoked and S.E.M. are presented. The number of basal release. Means culture dishes analyzed (derived from 3 to 5 cell preparations) is given in parentheses. All the differences shown were statistically significant. Other amino acids were not affected (Levi and Patrizio, 1992). a P < 0.0001. P < 0.01. P < 0.02. P = 0.05.
*
cluded by the experiments reporteL in Table IV, which show that the two agonists did not alter cell volume in either type-1 or type-2 astrocytes in the experimental conditions adopted. On the other hand, exposure to 50 mM KCl (replacing an equimolar concentration of NaCl) induced cell swelling (Table IV) and release of taurine > glutamate > aspartate (Table V) in both type-1 and type-2 astrocytes, in agreement with the above mentioned findings of other authors. The fact that the increase in cell volume was higher in type-2 than in type-1 astrocytes may explain the higher stimulation of taurine and glutamate release observed in type-2 astrocytes. Interestingly, the endogenous level of taurine and glutamate was substantially lower in type-2 as compared to type-1 astrocytes (Table 111). If taurine and glutamate are involved in cell volume regulation in astrocytes, the lower endogenous con-
centration of these amino acids in type-2 astrocytes might account for the greater propensity of these cells to swell. It is worth noting that a number of neutral amino acids whose endogenous level was high (glutamine, glycine, threonine, alanine) were not released by high [K+ ]-induced swelling. This suggests that the charge of the amino acid may be important in this type of release. On the other hand, some of the above mentioned neutral amino acids were released by non-NMDA receptor agonists in type-2 astrocytes. The differences in the release profiles observed in the two conditions provide evidence for the specificity of the release patterns observed, and indicate that the mechanisms underlying the release process are different in the two depolarizing conditions (non-NMDA receptor activation and high [K +]-induced swelling, respectively). By analogy with the results obtained when studying the mechanism of KA-induced [3H]GABA release from type-2 astrocytes (Gallo et al., 1991) it can be suggested that the release of endogenous amino acids induced by non-NMDA receptor agonists occurs through the membrane carriers operating in an outward direction when the intracellular “a+] is increased following receptor activation. On the other hand, swelling-induced release is unlikely to be related to carrier-mediated transport processes (Kimelberg et al., 1990).
Conclusions In conclusion, our results indicate that reasonably low concentrations of non-NMDA receptor agonists stimulate the release of exogenous [3H]GABA and [3H]~-aspartate, and of a group of endogenous amino acids from a defined subpopulation of astrocytes (type-2 astrocytes), without causing cell swelling. Some of the amino acids released are directly neuroactive (glutamate, taurine), some may influence the activity of NMDA receptors (glycine, see Ascher and Johnson, 1990). It is premature to suggest what could be the functional implications of this observation in the living brain. Another relevant aspect of our study is that type-2
249
astrocytes respond to high [K+] by a volume increase and by releasing three neuroactive amino acids similarly to type-1 astrocytes. This is important in view of the fact that the two astroglial populations belong to different cell lineages, and show a number of functional differences, as outlined in the first part of the present report. Acknowledgements
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