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K+- and temperature-evoked taurine efflux from hypothalamic astrocytes
Neuroscience Letters, 119 (1990) 23-26
23
Elsevier Scientific Publishers Ireland Ltd. NSL 07243
K +- and temperature-evoked taurine etflux from hypothalamic astrocytes G a r y A. Tigges 1, R o b e r t A. Philibert 1,2 and G a r y R. D u t t o n 1 1Department of Pharmacology and 2Departmentof Psychiatry, College of Medicine, University of Iowa, Iowa City, IA 52242 (U.S.A.) (Received 8 June 1990; Revised version received 27 June 1990; Accepted 4 July 1990)
Key words: Taurine; Temperature dependence; Astrocyte culture; Cerebellum; Hypothalamus Hypothalamic astrocytes in culture released taurine, a suspected inhibitory amino acid neurotransmitter/neuromodulator/osmoregulator, in response to isoosmotically increasing extracellular K + in a dose-dependent fashion. In the absence of added Ca 2+, basal release levels rose to approach those obtained after exposure to 60 mM K + in the presence of 2.5 mM Ca 2+, and were only partially lowered by the addition of 10 mM Mg 2+. Stimulation with K + (60 mM) did not further increase taurine etflux above the high basal levels seen in the absence of Ca 2+. Under standard conditions complete replacement of Na + with choline C1 had little effect on basal taurine release, but reduced K+-evoked (60 mM) efltux by 60%. The temperature dependence of the basal levels of taurine released from hypothalamic astroeytes was similar to that seen for cultured cerebellar astrocytes and neurons over the range 5-50°C. Taurine release increased from 5 to 15°C, remained constant between 15 and 33°C, decreased between 33 and 37°C and increased thereafter. The infection point of increased basal taurine release seen around 37°C (most prominent in astrocytes), may be of physiological significance. Results presented also show that the ion (Na +, Ca 2+ and K +) sensitivities of taurine etflux for cultured hypothalamic astrocytes are similar to those previously reported for cultured astrocytes from the cerebellum.
Taurine (2-aminoethanesulphonic acid) is distributed heterogeneously within the CNS reaching high concentrations in the hypothalamus, striatum, cerebellum and retina [6]. Evidence for the existence of taurinergic neurons includes the observation that taurine accumulates in specific synaptosomal populations different from those that accumulate ?-aminobutyric acid (GABA) and norepinephrine [10]. In addition, spontaneous and evoked release of taurine from superfused hypothalamic synaptosomal pellets has been detected [3]. However, its role as a neurotransmitter has still not been established [4]. Because of taurine's hypothalamic localization, one of the roles proposed for it has been in temperature regulation [5, 16]. Certain hypothalamic neurons, especially those in the preoptic region and anterior hypothalamus, are temperature-sensitive and alter their firing rates when the temperature in this region changes [9]. Intraventricularly injected taurine reduces body temperature in a variety of animal species, and a bilateral injection of taurine into the preoptic hypothalamus of the rat produces a dose-related hyperthermia, and hypothermia at very high doses [8]. In addition, microinfused and intraventricularly injected taurine can also interfere with the
Correspondence: G.R. Dutton, Department of Pharmacology, College of Medicine, The University of Iowa, Iowa City, IA 52242, U.S.A. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
hypothalamic control of hormonal release mechanisms [7, 15]. Although the neuronal origin and neuromodulatory role of taurine in the hypothalamus has been clearly demonstrated, the possibility that astrocytes in this region may also be a source of this amino acid has not been investigated. Thus, having previously demonstrated that taurine is released from cultured cerebellar astrocytes in a Ca 2+- and dose-dependent way in response to elevated K +, we set out to determine if hypothalamic astrocytes had similar properties. In addition, the Na + and temperature dependence of taurine release from cells was also investigated and compared to responses obtained from cerebellar astrocyte and neuronal cultures under identical conditions. Primary, dissociated hypothalamic and cerebellar neuronal and glial cultures were prepared and maintained as previously reported [1, 12]. Briefly, hypothalami (4 to 8-day-old Sprague-Dawley rats) or cerebella (7 to 9-day-old rats) were removed, enzymatically dissociated, triturated, and cells purified by centrifugation through 4 % bovine serum albumin. Neural cell suspensions were plated on 22 mm 2 poly-D-lysine-coated coverslips at a density of 0.5 x 106 cells/coverslip for glial cultures, and at 7.5 x 106 cells/coverslip for neuronenriched cultures. The glial culture medium consisted of Eagle's minimal essential medium supplemented with
24
10 % fetal calf serum, 33 mM glucose, 5.5 mM glutamine, and 180 p M gentamycin. One ml of fresh medium (of a total of 3 ml) was exchanged on the 4th day in vitro (DIV) and thereafter every 3rd day until use at 16-18 DIV. The neuronal culture medium contained, in addition to the components listed above, 23 m M K ÷ and 2.5% chick embryo extract. 5-Fluorodeoxyuridine, a mitotic inhibitor, was also added to the neuronal medium after 17 hours and the cells were used at 7-9 DIV. Two culture-bearing coverslips were placed in a special perfusion chamber and experiments performed as described in Pearce et al. [11]. The basic perfusion buffer consisted of 150.5 mM NaC1, 2.0 mM KC1, 2.5 mM CaC12, 1.0 mM MgSO4, 4.2 m M NaHCO3, 1 mM NaH2PO4 0.05 m M glutamine, and 12.5 mM HEPES (pH 7.4). The chamber was kept at 37°C in a water bath and the cultures were permitted to equilibrate for 25 min. In experiments where extracellular ionic concentrations were isoosmotically manipulated by altering the NaCI content, stimulations were carried out for 10 min after which the cultures were returned to the original perfusion conditions for 15 minutes. Basal efflux levels were defined as average efflux during the pre-stimulation period, and evoked release calculated by integrating peak areas of taurine released from a 10 min stimulation period [13]. In the temperature-dependence experiments, cultures were equilibrated at 37°C after which the temperature of the water bath was either decreased or increased (separate cultures) at a rate of 0.75°C/min during perfusion with low K + (2 mM) buffer. One ml perfusate fractions (2.5 min) were collected throughout. Samples were dried, redried, and derivitized and the endogenous taurine content measured via high performance liquid chromatography (HPLC) according to Rogers et al. [14]. Student's two-tailed t-test was used for statistical analysis with significance set at P < 0.05. Best
Standard conditions a
TABLE I
No added Ca 2+
K+-EVOKED TAURINE EFFLUX ASTROCYTES
FROM HYPOTHALAMIC
Numbers in parentheses represent independent experiments. See text for description of experimental paradigm and calculations. Equilibration Basal efflux buffer (con(pmol/min) trol)
Stimulation buffer
Stimulated efliux (pmol/ min)
% Increase above basal level
2 m M K+ (5) 119+24 2 m M K + (5) 89-+20 2 m M K+ (5) 112+34 2mMK+(5) 153+16
20mMK ÷ 40 m M K ÷ 60mMK + 80mMK ÷
145-+22 284-+ 17" 500+26* 1490-+3"
21 218 348 872
*P < 0.01 with respect to paired controls.
fit curves were generated using Sigmaplot (Jandel Scientific) and are third order polynomials (see Fig. 1). Increased taurine efflux from hypothalamic astrocytes in response to isoosmotic K + stimulation was dose-dependent over the range 20-80 mM, and increased above basal levels from about 21% at 20 m M to 872% at 80 mM K + without plateauing at higher levels (Table I). In the presence of 2.5 mM Ca 2+ basal efflux remained low, but increased markedly to 298 % above control values in the absence of added Ca 2+ (Table II). K+-induced taurine efflux was sensitive to the presence of Ca 2+ at 60 mM K +, in that changing the perfusion buffer from 2.5 mM Ca 2+ and 1.0 m M Mg 2+ to 0 m M Ca 2+ and 10 m M Mg 2÷ significantly reduced the K+-induced taurine efflux, and also raised basal efflux levels. Thus, efflux evoked by 60 m M K + was eliminated in the absence of extracellular Ca 2+ when compared to its paired basal control. K+-stimulated efflux appeared to be partially dependent on the presence of extracellular Na +. Although taurine efflux in response to 60 mM K + stimulation was not significantly diminished when only half the Na + was
T A B L E II E F F E C T S OF Ca 2÷ A N D Na ÷ O N K + - E V O K E D T A U R I N E EFFLUX FROM HYPOTHALAMIC ASTROCYTES "Standard conditions: 2 m M K ÷, 2.5 m M Ca 2÷, 150 m M Na +, I m M Mg2+. b Identical to equilibration buffer but with added K + (60 mM). N u m b e r s in parentheses represent independent experiments. See text for description of experimental paradigm and calculations. Equilibration Basal efflux buffer (con(pmol/min) trol)
Stimulation buffer b
Stimulated efflux (pmol/ min)
% Increase above basal level
112-+34
60 m M K ÷
500__+26*
348 (5)
4 4 6 + 18"*
60 m M K ÷
502__+46
13 (5)
No added Ca 2+ + 1 0 m M M g 2+ 284_+22**
60mMK ÷
319+31"
12(5)
75 m M Na + + 75 m M choline CI
60mMK ÷
493+30"
288 (5)
60mM K +
231 + 3 0 " * *
127+23
No added Na + + 150 m M choline C1 164 _ 14"*
40 (5)
*P<0.01 with respect to paired control. **P < 0.01 with respect to basal or stimulated etflux levels under standard conditions.
25
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CO .<
40.
40.
HY ASTROCYTES
40
CB NEURONS
30.
30-
CB ASTROCYTES
30
m Ix
2*0
20-
0
u
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10.
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2O TEMPERATURE
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50
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40 (*C)
.
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Fig. 1. Temperature-dependence of taurine etflux for cultured hypothalamic (HY) astrocytes compared to cerebellar (CB) neurons and astrocytes obtained from postnatal rats. Cultures were first equilibrated at 37°C, then the temperature was either decreased or increased at 0.75°C/min while perfusing with low K ÷ (2 mM) buffer (see text) at 0.4 ml/min. Data are from a representative experiment which were each performed a minimum of 5 times.
replaced by choline CI, it was reduced by 60% when Na + was completely replaced by choline CI (Table II). In temperature dependence experiments, hypothalamic astrocytes showed a slight decrease in taurine etflux as the temperature was lowered to about 33°C, while an increase in efflux occurred at temperatures above 37°C (Fig. 1). This was also seen for astrocyte and neuronal cerebellar cultures prepared from 7 to 9-day-old rats and grown for 17-19 and 7-9 days in vitro, respectively (Fig. 1). In all 3 culture types an increase in taurine efflux was seen around an inflection point of 37°C in the temperature range of 33-41°C. At extremes of temperature, basal taurine efflux either decreased (5-15°C) or substantially increased (40-50°C). The hypothalamus is one of the primary integrative and controlling structures in CNS-regulated thermoregulation, and endogenous taurine may be involved there as a neurotransmitter or neuromodulator. We previously demonstrated that taurine efflux from cultured cerebellar astrocytes is both Na +- and Ca2+-sensitive and can be induced in a dose-dependent fashion by isoosmotically increasing extracellular K + levels [12]. In this study, it has been shown that taurine efflux from cultured hypothalamic astrocytes is also stimulated by K + in a dose-dependent way and is sensitive to both Ca 2+ and Na + . Thus, it is possible that K+-evoked taurine release from hypothalamic astrocytes may be, at least in part, an osmoregulatory response to glial swelling as we have previously proposed for cerebellar astrocytes [2]. Additionally, taurine, once released, may then be free to act on local neurons as an inhibitory neuromodulator. The basal etttux of taurine from hypothalamic astrocytes appears to rise both above and below 37°C within the temperature range 33~1°C. While this increase is small, it may represent a true physiological response to
temperature variation which may cause changes in cell volume. Increases in basal taurine efflux between 33°C and 37°C, and again between 37°C and 41°C, were also seen for cerebellar astrocytes and to a slightly lesser extent in cerebellar neurons. Thus, these changes are not specific to the hypothalamus or to astrocytes. However, it appears that astrocytes and neurons can alter taurine efflux in a temperature-dependent fashion, which may serve a protective function. It is not clear from these studies whether these changes in taurine efflux levels with changing temperature are mediated by transport phenomena, since altered rates of efflux are seen both above and below 37°C and at temperature extremes. In conclusion, it has been shown that taurine release from hypothalamic astrocytes is both Na +- and Ca 2+sensitive, consistent with our previous findings with cerebellar astrocytes. In addition, taurine efflux from these cells may be temperature dependent over the critical range 33-41°C, and may serve to protect astrocytes, and perhaps neurons, from thermally induced osmotic changes. The authors would like to thank Kathryn Andrews for manuscript preparation. This work was supported by N I H Grant NS 20632 (G.R.D.). 1 Dutton, G.R., Currie, D.N. and Tear, K., An improved method for the bulk isolation of viable perikarya from postnatal cerebellum, J. Neurosci. Methods, 3 (1981) 421-427. 2 Dutton, G.R. and Philibert, R.A., Taurine release from cultured astrocytes. In G. Levi (Ed.), Differentiation and Functions of Glial Cells, A.R. Liss, 1990, pp. 235-241. 3 Hanretta, A.T. and Lombardini, J.B., Properties of spontaneous and evoked release of taurine from hypothalamic crude P2 synaptosomal preparations, Brain Res., 378 (1986) 205-215. 4 Hanretta, A.T. and Lombardini, J.B., Is taurine a hypothalamic neurotransmitter? A model of the differential uptake and compart-
26
5
6 7
8
9
10
I1 •
mentalization of taurine by neuronal and glial cell particles from the rat hypothalamus, Brain Res. Rev., 12 (1987) 167-201. Hruska, R.E., Thut, P.D., Huxtable, R.J. and Bressler, B.L., Hypothermia produced by taurine. In R. Huxtable and A. Barbeau (Eds.), Taurine, Raven, New Yrok, 1976, pp. 347-356. Huxtable, R.J. and Sebring, L.A., Towards a unifying theory for the actions of taurine, Trends Physiol. Sci., 7 (1986) 481-485. Ikuyama, S., Okajima, T., Kato, K. and Iboyashi, H., Effect of taurine on growth hormone and prolactin secretion in rats: possible interaction with opioid peptidergic system, Life Sci., 43 (1988) 807812. Kerwin, R.W. and Pycock, R.J., Role of taurine as a possible transmitter in the thermoregulatory pathways of the rat, J. Pharm. Pharmacol., 31 (1979) 455-470. Kobayashi, S., Temperature-sensitive neurons in the hypothalamus: a new hypothesis that they act as thermostats, not as transducers, Prog. Neurobiol., 32 (1989) 103-125. Lahdesmaki, P., Karppinen, A., Saarni, H. and Winter, R., Amino acids in the synaptic vesicle fraction from calf brain: content, uptake and metabolism, Brain Res., 138 (1977) 295-308. Pearce, B.R., Currie, D.N., Dutton, G.R., Hussey, R.E.G., Beale, R. and Pigott, R., A simple perfusion chamber for studying neuro-
12
13
14
15
16
transmitter release from cells maintained in monolayer culture, J. Neurosci. Methods, 3 (1981 ) 255-259. Philibert, R.A., Rogers, K.L., Allen, A.J. and Dutton, G.R., Dosedependent, K+-stimulated efflux of endogenous taurine from primary astrocyte cultures is Ca2+-dependent, J. Neurochem., 51 (1988) 122-126. Philibert, R.A. and Dutton, G.R., Dihydropyridines modulate K ÷evoked amino acid and adenosine release from cerebellar neuronal cultures, Neurosci. Lett., 102 (1989) 97-102. Rogers, K.L., Philibert, R.A,, Allen, A.J., Wilson, E.J. and Dutton, G.R., HPLC analysis of putative amino acid neurotransmitters released from primary cerebellar cultures, J. Neurosci. Methods, 22 (1987) 173-179. Scheibel, J., Elsasser, T. and Ondo, J.G., Stimulation of prolactin secretion by taurine, a neurally depressant amino acid, Neuroendocrinology, 30 (1980) 350-354. Sgaragli, G.P. and Palmi, M., The role and mechanism of action of taurine in mammalian thermoregulation. In. S,S. Oja, L. Ahter, P. Kontro and M.K. Paasonen, (Eds.), Taurine: Biological Actions and Clinical Perspectives, Alan R. Liss, New York, 1985, pp. 343357.
23
Elsevier Scientific Publishers Ireland Ltd. NSL 07243
K +- and temperature-evoked taurine etflux from hypothalamic astrocytes G a r y A. Tigges 1, R o b e r t A. Philibert 1,2 and G a r y R. D u t t o n 1 1Department of Pharmacology and 2Departmentof Psychiatry, College of Medicine, University of Iowa, Iowa City, IA 52242 (U.S.A.) (Received 8 June 1990; Revised version received 27 June 1990; Accepted 4 July 1990)
Key words: Taurine; Temperature dependence; Astrocyte culture; Cerebellum; Hypothalamus Hypothalamic astrocytes in culture released taurine, a suspected inhibitory amino acid neurotransmitter/neuromodulator/osmoregulator, in response to isoosmotically increasing extracellular K + in a dose-dependent fashion. In the absence of added Ca 2+, basal release levels rose to approach those obtained after exposure to 60 mM K + in the presence of 2.5 mM Ca 2+, and were only partially lowered by the addition of 10 mM Mg 2+. Stimulation with K + (60 mM) did not further increase taurine etflux above the high basal levels seen in the absence of Ca 2+. Under standard conditions complete replacement of Na + with choline C1 had little effect on basal taurine release, but reduced K+-evoked (60 mM) efltux by 60%. The temperature dependence of the basal levels of taurine released from hypothalamic astroeytes was similar to that seen for cultured cerebellar astrocytes and neurons over the range 5-50°C. Taurine release increased from 5 to 15°C, remained constant between 15 and 33°C, decreased between 33 and 37°C and increased thereafter. The infection point of increased basal taurine release seen around 37°C (most prominent in astrocytes), may be of physiological significance. Results presented also show that the ion (Na +, Ca 2+ and K +) sensitivities of taurine etflux for cultured hypothalamic astrocytes are similar to those previously reported for cultured astrocytes from the cerebellum.
Taurine (2-aminoethanesulphonic acid) is distributed heterogeneously within the CNS reaching high concentrations in the hypothalamus, striatum, cerebellum and retina [6]. Evidence for the existence of taurinergic neurons includes the observation that taurine accumulates in specific synaptosomal populations different from those that accumulate ?-aminobutyric acid (GABA) and norepinephrine [10]. In addition, spontaneous and evoked release of taurine from superfused hypothalamic synaptosomal pellets has been detected [3]. However, its role as a neurotransmitter has still not been established [4]. Because of taurine's hypothalamic localization, one of the roles proposed for it has been in temperature regulation [5, 16]. Certain hypothalamic neurons, especially those in the preoptic region and anterior hypothalamus, are temperature-sensitive and alter their firing rates when the temperature in this region changes [9]. Intraventricularly injected taurine reduces body temperature in a variety of animal species, and a bilateral injection of taurine into the preoptic hypothalamus of the rat produces a dose-related hyperthermia, and hypothermia at very high doses [8]. In addition, microinfused and intraventricularly injected taurine can also interfere with the
Correspondence: G.R. Dutton, Department of Pharmacology, College of Medicine, The University of Iowa, Iowa City, IA 52242, U.S.A. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
hypothalamic control of hormonal release mechanisms [7, 15]. Although the neuronal origin and neuromodulatory role of taurine in the hypothalamus has been clearly demonstrated, the possibility that astrocytes in this region may also be a source of this amino acid has not been investigated. Thus, having previously demonstrated that taurine is released from cultured cerebellar astrocytes in a Ca 2+- and dose-dependent way in response to elevated K +, we set out to determine if hypothalamic astrocytes had similar properties. In addition, the Na + and temperature dependence of taurine release from cells was also investigated and compared to responses obtained from cerebellar astrocyte and neuronal cultures under identical conditions. Primary, dissociated hypothalamic and cerebellar neuronal and glial cultures were prepared and maintained as previously reported [1, 12]. Briefly, hypothalami (4 to 8-day-old Sprague-Dawley rats) or cerebella (7 to 9-day-old rats) were removed, enzymatically dissociated, triturated, and cells purified by centrifugation through 4 % bovine serum albumin. Neural cell suspensions were plated on 22 mm 2 poly-D-lysine-coated coverslips at a density of 0.5 x 106 cells/coverslip for glial cultures, and at 7.5 x 106 cells/coverslip for neuronenriched cultures. The glial culture medium consisted of Eagle's minimal essential medium supplemented with
24
10 % fetal calf serum, 33 mM glucose, 5.5 mM glutamine, and 180 p M gentamycin. One ml of fresh medium (of a total of 3 ml) was exchanged on the 4th day in vitro (DIV) and thereafter every 3rd day until use at 16-18 DIV. The neuronal culture medium contained, in addition to the components listed above, 23 m M K ÷ and 2.5% chick embryo extract. 5-Fluorodeoxyuridine, a mitotic inhibitor, was also added to the neuronal medium after 17 hours and the cells were used at 7-9 DIV. Two culture-bearing coverslips were placed in a special perfusion chamber and experiments performed as described in Pearce et al. [11]. The basic perfusion buffer consisted of 150.5 mM NaC1, 2.0 mM KC1, 2.5 mM CaC12, 1.0 mM MgSO4, 4.2 m M NaHCO3, 1 mM NaH2PO4 0.05 m M glutamine, and 12.5 mM HEPES (pH 7.4). The chamber was kept at 37°C in a water bath and the cultures were permitted to equilibrate for 25 min. In experiments where extracellular ionic concentrations were isoosmotically manipulated by altering the NaCI content, stimulations were carried out for 10 min after which the cultures were returned to the original perfusion conditions for 15 minutes. Basal efflux levels were defined as average efflux during the pre-stimulation period, and evoked release calculated by integrating peak areas of taurine released from a 10 min stimulation period [13]. In the temperature-dependence experiments, cultures were equilibrated at 37°C after which the temperature of the water bath was either decreased or increased (separate cultures) at a rate of 0.75°C/min during perfusion with low K + (2 mM) buffer. One ml perfusate fractions (2.5 min) were collected throughout. Samples were dried, redried, and derivitized and the endogenous taurine content measured via high performance liquid chromatography (HPLC) according to Rogers et al. [14]. Student's two-tailed t-test was used for statistical analysis with significance set at P < 0.05. Best
Standard conditions a
TABLE I
No added Ca 2+
K+-EVOKED TAURINE EFFLUX ASTROCYTES
FROM HYPOTHALAMIC
Numbers in parentheses represent independent experiments. See text for description of experimental paradigm and calculations. Equilibration Basal efflux buffer (con(pmol/min) trol)
Stimulation buffer
Stimulated efliux (pmol/ min)
% Increase above basal level
2 m M K+ (5) 119+24 2 m M K + (5) 89-+20 2 m M K+ (5) 112+34 2mMK+(5) 153+16
20mMK ÷ 40 m M K ÷ 60mMK + 80mMK ÷
145-+22 284-+ 17" 500+26* 1490-+3"
21 218 348 872
*P < 0.01 with respect to paired controls.
fit curves were generated using Sigmaplot (Jandel Scientific) and are third order polynomials (see Fig. 1). Increased taurine efflux from hypothalamic astrocytes in response to isoosmotic K + stimulation was dose-dependent over the range 20-80 mM, and increased above basal levels from about 21% at 20 m M to 872% at 80 mM K + without plateauing at higher levels (Table I). In the presence of 2.5 mM Ca 2+ basal efflux remained low, but increased markedly to 298 % above control values in the absence of added Ca 2+ (Table II). K+-induced taurine efflux was sensitive to the presence of Ca 2+ at 60 mM K +, in that changing the perfusion buffer from 2.5 mM Ca 2+ and 1.0 m M Mg 2+ to 0 m M Ca 2+ and 10 m M Mg 2÷ significantly reduced the K+-induced taurine efflux, and also raised basal efflux levels. Thus, efflux evoked by 60 m M K + was eliminated in the absence of extracellular Ca 2+ when compared to its paired basal control. K+-stimulated efflux appeared to be partially dependent on the presence of extracellular Na +. Although taurine efflux in response to 60 mM K + stimulation was not significantly diminished when only half the Na + was
T A B L E II E F F E C T S OF Ca 2÷ A N D Na ÷ O N K + - E V O K E D T A U R I N E EFFLUX FROM HYPOTHALAMIC ASTROCYTES "Standard conditions: 2 m M K ÷, 2.5 m M Ca 2÷, 150 m M Na +, I m M Mg2+. b Identical to equilibration buffer but with added K + (60 mM). N u m b e r s in parentheses represent independent experiments. See text for description of experimental paradigm and calculations. Equilibration Basal efflux buffer (con(pmol/min) trol)
Stimulation buffer b
Stimulated efflux (pmol/ min)
% Increase above basal level
112-+34
60 m M K ÷
500__+26*
348 (5)
4 4 6 + 18"*
60 m M K ÷
502__+46
13 (5)
No added Ca 2+ + 1 0 m M M g 2+ 284_+22**
60mMK ÷
319+31"
12(5)
75 m M Na + + 75 m M choline CI
60mMK ÷
493+30"
288 (5)
60mM K +
231 + 3 0 " * *
127+23
No added Na + + 150 m M choline C1 164 _ 14"*
40 (5)
*P<0.01 with respect to paired control. **P < 0.01 with respect to basal or stimulated etflux levels under standard conditions.
25
LL U* t~J
CO .<
40.
40.
HY ASTROCYTES
40
CB NEURONS
30.
30-
CB ASTROCYTES
30
m Ix
2*0
20-
0
u
_z
I0.
10.
0-
O.
-I0
-
0
tO
2O TEMPERATURE
3O
40 (°C)
50
-10
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0
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10
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10 0 1
.
20
30
TEMPERATURE
_
.
40 (*C)
.
.
50
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20 TEMPERATURE
.
30
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Fig. 1. Temperature-dependence of taurine etflux for cultured hypothalamic (HY) astrocytes compared to cerebellar (CB) neurons and astrocytes obtained from postnatal rats. Cultures were first equilibrated at 37°C, then the temperature was either decreased or increased at 0.75°C/min while perfusing with low K ÷ (2 mM) buffer (see text) at 0.4 ml/min. Data are from a representative experiment which were each performed a minimum of 5 times.
replaced by choline CI, it was reduced by 60% when Na + was completely replaced by choline CI (Table II). In temperature dependence experiments, hypothalamic astrocytes showed a slight decrease in taurine etflux as the temperature was lowered to about 33°C, while an increase in efflux occurred at temperatures above 37°C (Fig. 1). This was also seen for astrocyte and neuronal cerebellar cultures prepared from 7 to 9-day-old rats and grown for 17-19 and 7-9 days in vitro, respectively (Fig. 1). In all 3 culture types an increase in taurine efflux was seen around an inflection point of 37°C in the temperature range of 33-41°C. At extremes of temperature, basal taurine efflux either decreased (5-15°C) or substantially increased (40-50°C). The hypothalamus is one of the primary integrative and controlling structures in CNS-regulated thermoregulation, and endogenous taurine may be involved there as a neurotransmitter or neuromodulator. We previously demonstrated that taurine efflux from cultured cerebellar astrocytes is both Na +- and Ca2+-sensitive and can be induced in a dose-dependent fashion by isoosmotically increasing extracellular K + levels [12]. In this study, it has been shown that taurine efflux from cultured hypothalamic astrocytes is also stimulated by K + in a dose-dependent way and is sensitive to both Ca 2+ and Na + . Thus, it is possible that K+-evoked taurine release from hypothalamic astrocytes may be, at least in part, an osmoregulatory response to glial swelling as we have previously proposed for cerebellar astrocytes [2]. Additionally, taurine, once released, may then be free to act on local neurons as an inhibitory neuromodulator. The basal etttux of taurine from hypothalamic astrocytes appears to rise both above and below 37°C within the temperature range 33~1°C. While this increase is small, it may represent a true physiological response to
temperature variation which may cause changes in cell volume. Increases in basal taurine efflux between 33°C and 37°C, and again between 37°C and 41°C, were also seen for cerebellar astrocytes and to a slightly lesser extent in cerebellar neurons. Thus, these changes are not specific to the hypothalamus or to astrocytes. However, it appears that astrocytes and neurons can alter taurine efflux in a temperature-dependent fashion, which may serve a protective function. It is not clear from these studies whether these changes in taurine efflux levels with changing temperature are mediated by transport phenomena, since altered rates of efflux are seen both above and below 37°C and at temperature extremes. In conclusion, it has been shown that taurine release from hypothalamic astrocytes is both Na +- and Ca 2+sensitive, consistent with our previous findings with cerebellar astrocytes. In addition, taurine efflux from these cells may be temperature dependent over the critical range 33-41°C, and may serve to protect astrocytes, and perhaps neurons, from thermally induced osmotic changes. The authors would like to thank Kathryn Andrews for manuscript preparation. This work was supported by N I H Grant NS 20632 (G.R.D.). 1 Dutton, G.R., Currie, D.N. and Tear, K., An improved method for the bulk isolation of viable perikarya from postnatal cerebellum, J. Neurosci. Methods, 3 (1981) 421-427. 2 Dutton, G.R. and Philibert, R.A., Taurine release from cultured astrocytes. In G. Levi (Ed.), Differentiation and Functions of Glial Cells, A.R. Liss, 1990, pp. 235-241. 3 Hanretta, A.T. and Lombardini, J.B., Properties of spontaneous and evoked release of taurine from hypothalamic crude P2 synaptosomal preparations, Brain Res., 378 (1986) 205-215. 4 Hanretta, A.T. and Lombardini, J.B., Is taurine a hypothalamic neurotransmitter? A model of the differential uptake and compart-
26
5
6 7
8
9
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