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Glutamate receptor agonists increase intracellular Ca2+ independently of voltage-gated Ca2+ channels in rat cerebellar granule cells
Neuroscience Letters, 98 (1989) 57 62 Elsevier Scientific Publishers Ireland Ltd.
57
NSL 05919
Glutamate receptor agonists increase intracellular Ca 2 + independently of voltage-gated Ca 2 + channels in rat cerebellar granule cells I. H o l o p a i n e n 1"2, M . O . K . Enkvist 2 and K . E . O . ,~kerman 2 II)epartment ~71'Biomedical Sciences, University o/" Tampere, Tarnpere (Finland), Department ol Phr.~'io/o~,,v. UHiversiO" qf Turku. Turku (Finland) and eDepartment of Biochemistry and Pharmac.v, ,4ho Akademi, Turku (Finland) (Received 8 August 1988: Revised version received 2 November 1988; Accepted 4 November 1988)
Key word,~" Cerebellar granule cell; Excitatory amino acid; Fura-2; Bisoxonol: Membrane potential: Intracellular calcium Changes in membrane potential and cytosolic free Ca 2+ concentrations, [Ca t ~]~, in response to l.-glutamate and glutamate receptor agonists were measured in rat cerebellar granule cells grown on coverslips. The membrane was depolarized by the application of L-glutamate and kainate, and by elevating the extracellular K + concentration, as determined by using the membrane potential probe bisoxonol (DiBA-C4(3)). The [Cae']~ as measured with fura-2 was 220 nM on average under resting conditions and increased by raising the extracellular K + and by applying L-glutamate, kainate, quisqualate or N-methyl-D-aspartate (NMDA). Verapamil and nifedipine reduced the high-K + induced rise in [Ca:+]~ but did not significantly affect the responses produced by N M D A , quisqualate and kainate, suggesting that the increase in intracellular Ca 2~ in response to glutamate receptor agonists is primarily due to Ca -'+ influx through receptorcoupled ion channels.
The excitatory amino acid L-glutamate acts as a neurotransmitter in the cerebellar granule cells, which excite interneurons and Purkinje cells [3, 14]. The activation of the excitatory amino acid receptors in various cerebellar preparations has been shown to lead to an increase in Ca 2~ influx [16], turnover of inositol phospholipids [12], and changes in the levels of cyclic nucleotides [5, 13]. Recent experiments suggest that cultured cerebellar granule cells possess all three main subtypes of the glutamate receptor [8, 16]. Measurements based on injected arsenazo III in cultured spinal cord neurons have suggested that the N M D A subtype is primarily linked to Ca :+ influx [11]. An increase in cytosolic Ca 2+ upon application of kainate has, however, recently been observed in single cell recordings of cultured cerebellar granule cells [2]. Whether the rise in intracellular Ca :+ occurred as a result of Ca 2~- influx through Correspondence: 1. Holopainen. Present address: Department of Physiology, University of Turku, Kiinamyllynk 10, SF-20500 Turku. Finland. 0304-3940 ,~9 $ ~.50 ~ 198q Elsevier Scientific Publishers Ireland Lid.
58 voltage-dependent, or receptor-linked channels, or whether it was due to release from internal stores, has remained undetermined so far. The aim of the present study was to clarify the mechanism of the changes in intracellular Ca 2+ induced by L-glutamate and its agonists in cerebellar granule cells grown on coverslips using the fura-2 method [4, 15]. Granule cells were obtained from the cerebella of 7-day-old Wistar rats and cultured for 7-9 days on coverslips coated with poly-L-lysine in tissue culture dishes as described in detail elsewhere [9]. The fluorochromes were obtained from Molecular Probes Inc. (Junction City, U.S.A.). For the fluorescence recordings coverslips were removed from the culture dishes, carefully rinsed with a balanced salt solution (BSS; composition in raM: NaC1 137, KC1 5.0, CaCI2 1.0, KH2PO4 0.44, NaHCO3 4.2, 2-(3hydroxyl-i,l- bis(hydroxymethyl)ethyl)aminoethanesulphonate (TES) 20, (pH 7.4, 37°C) and mounted in a temperature-controlled cuvette (37°C) containing 2.0 ml of the BSS medium with mechanical stirring. A portion of 0.5/11 from the bisoxonol (bis(l,3-dibutylbarbituric acid)trimethineoxonol), DiBA-C4-(3) stock solution (0.4 raM, prepared in dimethyisulfoxide, DMSO) [1] was added directly into the cuvette containing the coverslip and the fluorescence (495 nm ex., 517 nm em.) was recorded alter stabilization of the signal after about 20 min using a Hitachi F 4000 fluorescence spectrofotometer. For the determination of intracellular free Ca 2+, 10/11 of the acetoxymethyl ester of fura-2 (fura-2/AM) dissolved in DMSO (2 mg/ml) was added to the dishes containing 5 ml of the culture medium. The dishes were subsequently incubated for 40 min at 37°C in an air ventilated incubator. For the fluorescence recordings coverslips were carefully rinsed with BSS solution and thereafter mounted in the temperature-controlled cuvette (37°C) containing 2.0 ml of BSS medium supplemented with 0.5% bovine serum albumin. A magnetic stirrer was used to continuously mix the sample to ascertain the rapid and even distribution of the added compounds. The fluorescence at 340 nm (ex.) and 505 nm (era.) was then recorded. For calibration, maximal fluorescence levels were obtained by adding 1.0 ~M ionomycin and the fluorescence minimum by a subsequent addition of 0.1 mM MnCI2 [7]. A Kd value of 220 nM was used for the fura-2-Ca :+ complex [4], and calculation of the free Ca 2+ concentration was performed as in Tsien et al. [15]. Changes in DiBA-C4-(3) fluorescence in response to additions of glutamate and kainate are shown in Fig. I. Glutamate increased the fluorescence of the probe indicating a depolarization of the cells (Fig. IA). A subsequent addition of kainate did not significantly increase the fluorescence. In order to get information concerning the magnitude of the potential changes the extracellular K ÷ concentration was subsequently increased stepwisely to 40 mM. Thereafter, the fluorescence of the bisoxonol. in response to similar increases in the K + concentration in the absence of glutamate, was recorded after the same coverslip had been rinsed in BSS medium at 37'~C for 30 min. The results are replotted in Fig. IC as a function of the apparent K + equilibrium potential (assuming the intracellular K + concentration is 100 mM) on the same scale as the trace in Fig. 1A. The responses in Fig. I A, C suggest that the depolarization caused by glutamate is of the order of 30 mV. An increase in bisoxonol fluorescence upon addition of kainate, when added after subsequent rinsing for 30
59
C
A
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i ZO
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2 min
B
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kd
W
Io~
/'-
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i
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t KA
Fig. I. Glutamate-, kainate-, and high-K ÷-induced changes in membrane potential as measured with the fluorescence DiBA-C4-(3),(bisoxonol) in cultured cerebellar granule cells. The recordings were made as described in the text. In (A) additions of glutamate (GLU, 0.5 mM), kainate (KA, 0.5 mM) and a stepwise increase in extracellular K + (K ÷) were made as indicated by the arrows. The scale is given as percent increase in bisoxonol fluorescence from the basal level. In B, subsequent additions of KA (0.5 mM) and GLU (0.5 mM) were made after rinsing the coverslip for 30 min in the BSS medium. The scale indicates the percent increase in fluorescence from the basal level. In C, the same coverslip as in A was rinsed for 30 rain in BSS medium and the fluorescencein response to increased external K ÷ (5~,0 mM) was recorded. The increase in bisoxonol fluorescence is plotted as a function of the apparent K + equilibrium potential (calculated from the Nernst equation; EK-+ -- 59 × log [K +]i,/[K+]out)assuming an internal K + concentration of I00 mM. In the y-axis 100% indicates the increase in the fluorescence from the basal level to that in the presence of 40 mM K +. Original recordings are retraced (A, B).
m i n in BSS m e d i u m is shown in Fig. lB. A n a p p l i c a t i o n of g l u t a m a t e after kainate caused only a small further increase in fluorescence. The lack of additivity in the responses suggest that b o t h agonists depolarize cells by a similar m e c h a n i s m . C h a n g e s in intracellular free Ca 2+ as m e a s u r e d with fura-2 are shown in Fig. 2. The basal intracellular Ca 2+ was 217 +_21 n M (n = 23). A n increase in the K + concent r a t i o n from 5 to 25 m M resulted in a rapid increase in the intensity o f the fluorescence i n d i c a t i n g a rise in [Ca2+]i to a value of a b o u t 600 n M (Fig. 2A). This peak decayed within 1 m i n to a plateau. A similar transient response to K + has recently been observed in single cell recordings by using fura-2 in cerebellar g r a n u l e cells in explant cultures [2]. Since the increase in [Ca2+]i was n o t seen when 2 m M E G T A was added immediately prior to the a d d i t i o n of K + (data not shown), it most p r o b a bly represents a flow o f Ca 2+ into cells. The response to the a d d i t i o n of K ÷ was considerably reduced in the presence of verapamil (0.1 m M ) (Fig. 2B) a n d nifedipine (50 /tM) (Fig. 2C), blockers of voltage-gated Ca 2+ channels, i n d i c a t i n g that the rise in [Ca2+]i was due to Ca 2+ entry t h r o u g h v o l t a g e - d e p e n d e n t Ca 2+ c h a n n e l s opened by depolarization. K a i n a t e caused a sustained increase in the intracellular Ca 2+ concentration (Fig. 2D). Verapamil (0. I raM, in 6 experiments out of 6) (Fig. 2E) a n d nifedi-
60
A
Cai(nM)
B
Cai (riM)
Cai (riM) ~
fI ~
t K"
D
r290 L260
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K÷
E
Cai [riM)
~1 KA
~| KA
230
L183
Cai(nM)
F
L2L0
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t KA
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Cai{nM}
H "
~50
°
i,.,,l NMDA
U
230
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180
NMDA
Fig. 2. Effects of verapamil and nifedipine on high-K +- and glutamate receptor agonists-induced changes in [Ca2+]i as measured with fura-2 in cultured cerebellar granule cells. The cells were loaded with fura-2 and recordings done as described in the text. In A, addition of K + (25 raM) was made as indicated by the arrow. In B and C, verapamil (0.l mM) and nifedipine (50 #M), respectively, were added 2 min prior to the addition of K + (25 mM). In D, application of KA (0.5 mM) was made as indicated, and in E and F, verapamil (0.1 mM) and nifedipine (50 #M), respectively, were added 2 min prior to KA (0.5 mM). In G, NMDA (0.25 mM) and quisqualate (QU, 0.25 raM) were added as indicated. In H, nifedipine (50 #M) was added 2 rain prior to NMDA (0.25 mM) and QU (0.25 raM). Original recordings are retraced.
pine (50 #M in 3 experiments out of 3) (Fig. 2F) had no significant effect on the kainate-induced rise in fura-2 fluorescence. Sequential additions of N M D A and quisqualate also caused increases in fluorescence (Fig. 2G), which were not significantly reduced in the presence of nifedipine (50 MM) (Fig. 2H). These results demonstrate that the mechanism of the rise in [Ca2+]i is different from that induced by high-K, and is not a result of depolarization-linked opening of voltage-gated Ca 2~ channels. The stepwise rise in cytosolic Ca 2+ by sequential additions of NMDA, quisqualate and kainate (Fig. 3A) was totally inhibited by a prior addition of EGTA (2 mM) (3B). Fig. 3C shows the dose response curves for the increase in fura-2 fluorescence by the agonists. Glutamate was the most effective in increasing cytosolic Ca 2+ with an EDs0 value around 5 #M in keeping with the data obtained in isolated hippocampal neurons [10]. The EDs0 values of the other agonists were 10 100 times higher than
61
A
100~
Cai (nM)
C
Fis°°
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C
-6 50
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NMDA
x I
I
1 rain
B
/ NMDA
....__
1
t
OU
KA
r L
~ 9s
0
I
0.1
10 Agonist (pM)
I
1000
Fig. 3. Effects of N M D A , Q U and K A on [Ca-'+]i in cultured cerebellar granule cells as measured with fura-2. The recordings were carried out as in Fig. 2. In A, subsequent additions of N M D A ((I.5 mMt. QU (0.25 m M ) and KA (0.5 m M ) were made as indicated by the arrows. In B, E G T A (2.0 mM) was added immcdiately prior to N M D A (0.25 mM), Q U (0.25 m M ) and KA (0.25 mM). In C, the dose response curves lk~r G LU (O), KA (A), Q U (!~) and N M D A ( O ) are plotted as per cent of maximal fluorescence signal obtained by the addition o f i o n o m y c i n (1.0 ItM).
that of glutamate. N M D A had the smallest effect. The maximal response obtained with this agonist was only about 30% of that obtained with glutamate. The present results suggest that the glutamate receptor agonist-induced increase in intracellular Ca 9+ in granule cells is primarily due to Ca 2+ entry through Ca -~+permeable receptor coupled cation channels. The lack of an effect of glutamate receptor agonists in the absence of extracellular Ca 2+ as well as the insensitivity to verapamil and nifedipine are in agreement with this. Thus the increase in phosphatidylinositol turnover and gyanylate cyclase activation in response to glutamate [12, 13] might be the result of the increased Ca 2~ concentration as is the case with synaptoneurosomes [6]. Our results also show that cerebellar granule cells react to all three glutamate receptor agonists, kainate, quisqualate and N M D A with a rise in cytosolic Ca 2+ in contrast to spinal cord neurones [11], in which cells mainly the N M D A receptors seem to be linked to Ca 2+ influx. This study was aided by grants from the Academy of Finland, the Sigrid Jusdlius foundation and the Borg foundation. We thank Mr. Pekka Ketola for expert construction of equipment needed during this study. I Br~iuncr, T., Hfilster, D.F. and Strasser, R.J., Comparative measurements of membrane potentials with microelectrodes and voltage-sensitive dyes, Biochim. Biophys. Acta, 771 (1984) 208 216. 2 Connor, J.A., Tseng, H.-Y. and Hockberger, P.E., Depolarization- and transmitter-induced changes in intracellular Ca: + of rat cerebellar granule cells in explant cultures, J. Neurosci., 7 ( 19871 1384 1400. 3 Dupont, J.-L., Fournier, E., Gardette, R. and Crepel, F., Effect of excitatory amino acids on Purkinjc cell dendrites in cerebellar slices from normal and staggerer mice, Neuroscience, 12 (19841 613 6[9. 4 Grynkiewicz. G., Poenie, M. and Tsien, R.Y., A new generation of Ca 2+ indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3440 3450.
62 5 Garthwaite, J. and Garthwaite, G., Cellular origins of cyclic GMP responses to excitatory amino acid receptor agonists in rat cerebellum in vitro, J. Neurochem., 48 (1987) 29 39. 6 Gusovsky, F., Hollingsworth, E.B. and Daly, J.W., Regulation of phosphatidylinositol turnover in brain synaptoneurosomes: stimulatory effects of agents that enhance influx of sodium ions. Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 3003 3007. 7 Hesketh, T.R., Smith, G.A., Moore, J.P., Taylor, M.V. and Metcalfe, J.C., Free cytoplasmic calcium concentration and the mitogenic stimulation of lymphocytes, J. Biol. Chem., 258 (1983) 4876-4882. 8 Holopainen, I. and Kontro, P., Glutamate release from cerebellar granule cells differentiating in culture: modulation of the K+-stimulated release by inhibitory amino acids, Neurochem. lnt., 12 (1988) 155 161. 9 Holopainen, I., Malminen, O, and Kontro, P., Sodium-dependent high-affinity uptake of taurine in cultured cerebellar granule cells and astrocytes, J. Neurosci. Res., 18 (1987) 479483. 10 Kudo, Y. and Ogura, A., Glutamate-induced increase in intracellular Ca 2+ concentration in isolated hippocampal neurones, Br. J. Pharmacol., 89 (1986) 191 -198. I I MacDermott, A.B., Mayer, M.L., Westbrook, G.L., Smith, S.J. and Barker, J.L., NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones, Nature (Lond.), 321 (1986) 519-522. 12 Nicoletti, F., Wroblewski, J.T., Novelli, A., Alho, H., Guidotti, A. and Costa, E,, The activation of inositol phospholipid metabolism as a signal transducing system for dicarboxylic excitatory amino acids in primary cultures of cerebellar granule cells, J. Neurosci., 6 (1986) 190:%1911. 13 Novelli, A., Nicoletti, F., Wroblewski, J.Y., Alho, H., Costa, E. and Guidotti, A., Excitatory amino acid receptors coupled with guanylate cyclase in primary cultures of cerebellar granule cells, J. Neurosci., 7 (1987) 40-47. 14 Sandoval, M.E. and Cotman, C.W., Evaluation of glutamate as a neurotransmitter of cerebellar parallel fibers, Neuroscience, 3 (1978) 199-206. 15 Tsien, R.Y., Pozzan, T. and Rink, T.J., Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator, J. Cell Biol., 94 (1982) 325 334. 16 Wroblewski, J.T., Nicoletti, F. and Costa, E., Different coupling of excitatory amino acid receptors with Ca 2 ~ channels in primary cultures of cerebellar granule cells, Neuropharmacology, 24 (l 985) 919 921.
57
NSL 05919
Glutamate receptor agonists increase intracellular Ca 2 + independently of voltage-gated Ca 2 + channels in rat cerebellar granule cells I. H o l o p a i n e n 1"2, M . O . K . Enkvist 2 and K . E . O . ,~kerman 2 II)epartment ~71'Biomedical Sciences, University o/" Tampere, Tarnpere (Finland), Department ol Phr.~'io/o~,,v. UHiversiO" qf Turku. Turku (Finland) and eDepartment of Biochemistry and Pharmac.v, ,4ho Akademi, Turku (Finland) (Received 8 August 1988: Revised version received 2 November 1988; Accepted 4 November 1988)
Key word,~" Cerebellar granule cell; Excitatory amino acid; Fura-2; Bisoxonol: Membrane potential: Intracellular calcium Changes in membrane potential and cytosolic free Ca 2+ concentrations, [Ca t ~]~, in response to l.-glutamate and glutamate receptor agonists were measured in rat cerebellar granule cells grown on coverslips. The membrane was depolarized by the application of L-glutamate and kainate, and by elevating the extracellular K + concentration, as determined by using the membrane potential probe bisoxonol (DiBA-C4(3)). The [Cae']~ as measured with fura-2 was 220 nM on average under resting conditions and increased by raising the extracellular K + and by applying L-glutamate, kainate, quisqualate or N-methyl-D-aspartate (NMDA). Verapamil and nifedipine reduced the high-K + induced rise in [Ca:+]~ but did not significantly affect the responses produced by N M D A , quisqualate and kainate, suggesting that the increase in intracellular Ca 2~ in response to glutamate receptor agonists is primarily due to Ca -'+ influx through receptorcoupled ion channels.
The excitatory amino acid L-glutamate acts as a neurotransmitter in the cerebellar granule cells, which excite interneurons and Purkinje cells [3, 14]. The activation of the excitatory amino acid receptors in various cerebellar preparations has been shown to lead to an increase in Ca 2~ influx [16], turnover of inositol phospholipids [12], and changes in the levels of cyclic nucleotides [5, 13]. Recent experiments suggest that cultured cerebellar granule cells possess all three main subtypes of the glutamate receptor [8, 16]. Measurements based on injected arsenazo III in cultured spinal cord neurons have suggested that the N M D A subtype is primarily linked to Ca :+ influx [11]. An increase in cytosolic Ca 2+ upon application of kainate has, however, recently been observed in single cell recordings of cultured cerebellar granule cells [2]. Whether the rise in intracellular Ca :+ occurred as a result of Ca 2~- influx through Correspondence: 1. Holopainen. Present address: Department of Physiology, University of Turku, Kiinamyllynk 10, SF-20500 Turku. Finland. 0304-3940 ,~9 $ ~.50 ~ 198q Elsevier Scientific Publishers Ireland Lid.
58 voltage-dependent, or receptor-linked channels, or whether it was due to release from internal stores, has remained undetermined so far. The aim of the present study was to clarify the mechanism of the changes in intracellular Ca 2+ induced by L-glutamate and its agonists in cerebellar granule cells grown on coverslips using the fura-2 method [4, 15]. Granule cells were obtained from the cerebella of 7-day-old Wistar rats and cultured for 7-9 days on coverslips coated with poly-L-lysine in tissue culture dishes as described in detail elsewhere [9]. The fluorochromes were obtained from Molecular Probes Inc. (Junction City, U.S.A.). For the fluorescence recordings coverslips were removed from the culture dishes, carefully rinsed with a balanced salt solution (BSS; composition in raM: NaC1 137, KC1 5.0, CaCI2 1.0, KH2PO4 0.44, NaHCO3 4.2, 2-(3hydroxyl-i,l- bis(hydroxymethyl)ethyl)aminoethanesulphonate (TES) 20, (pH 7.4, 37°C) and mounted in a temperature-controlled cuvette (37°C) containing 2.0 ml of the BSS medium with mechanical stirring. A portion of 0.5/11 from the bisoxonol (bis(l,3-dibutylbarbituric acid)trimethineoxonol), DiBA-C4-(3) stock solution (0.4 raM, prepared in dimethyisulfoxide, DMSO) [1] was added directly into the cuvette containing the coverslip and the fluorescence (495 nm ex., 517 nm em.) was recorded alter stabilization of the signal after about 20 min using a Hitachi F 4000 fluorescence spectrofotometer. For the determination of intracellular free Ca 2+, 10/11 of the acetoxymethyl ester of fura-2 (fura-2/AM) dissolved in DMSO (2 mg/ml) was added to the dishes containing 5 ml of the culture medium. The dishes were subsequently incubated for 40 min at 37°C in an air ventilated incubator. For the fluorescence recordings coverslips were carefully rinsed with BSS solution and thereafter mounted in the temperature-controlled cuvette (37°C) containing 2.0 ml of BSS medium supplemented with 0.5% bovine serum albumin. A magnetic stirrer was used to continuously mix the sample to ascertain the rapid and even distribution of the added compounds. The fluorescence at 340 nm (ex.) and 505 nm (era.) was then recorded. For calibration, maximal fluorescence levels were obtained by adding 1.0 ~M ionomycin and the fluorescence minimum by a subsequent addition of 0.1 mM MnCI2 [7]. A Kd value of 220 nM was used for the fura-2-Ca :+ complex [4], and calculation of the free Ca 2+ concentration was performed as in Tsien et al. [15]. Changes in DiBA-C4-(3) fluorescence in response to additions of glutamate and kainate are shown in Fig. I. Glutamate increased the fluorescence of the probe indicating a depolarization of the cells (Fig. IA). A subsequent addition of kainate did not significantly increase the fluorescence. In order to get information concerning the magnitude of the potential changes the extracellular K ÷ concentration was subsequently increased stepwisely to 40 mM. Thereafter, the fluorescence of the bisoxonol. in response to similar increases in the K + concentration in the absence of glutamate, was recorded after the same coverslip had been rinsed in BSS medium at 37'~C for 30 min. The results are replotted in Fig. IC as a function of the apparent K + equilibrium potential (assuming the intracellular K + concentration is 100 mM) on the same scale as the trace in Fig. 1A. The responses in Fig. I A, C suggest that the depolarization caused by glutamate is of the order of 30 mV. An increase in bisoxonol fluorescence upon addition of kainate, when added after subsequent rinsing for 30
59
C
A
K + ,10 I zu ~ , ~
1Or
/" ./"
L//i 80 60
GLU
i ZO
1 20
A EK+(mV)
ta_
2 min
B
Z
Ioo r 50
0~-- 1
O D
~z.o
kd
W
Io~
/'-
o
i
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t KA
Fig. I. Glutamate-, kainate-, and high-K ÷-induced changes in membrane potential as measured with the fluorescence DiBA-C4-(3),(bisoxonol) in cultured cerebellar granule cells. The recordings were made as described in the text. In (A) additions of glutamate (GLU, 0.5 mM), kainate (KA, 0.5 mM) and a stepwise increase in extracellular K + (K ÷) were made as indicated by the arrows. The scale is given as percent increase in bisoxonol fluorescence from the basal level. In B, subsequent additions of KA (0.5 mM) and GLU (0.5 mM) were made after rinsing the coverslip for 30 min in the BSS medium. The scale indicates the percent increase in fluorescence from the basal level. In C, the same coverslip as in A was rinsed for 30 rain in BSS medium and the fluorescencein response to increased external K ÷ (5~,0 mM) was recorded. The increase in bisoxonol fluorescence is plotted as a function of the apparent K + equilibrium potential (calculated from the Nernst equation; EK-+ -- 59 × log [K +]i,/[K+]out)assuming an internal K + concentration of I00 mM. In the y-axis 100% indicates the increase in the fluorescence from the basal level to that in the presence of 40 mM K +. Original recordings are retraced (A, B).
m i n in BSS m e d i u m is shown in Fig. lB. A n a p p l i c a t i o n of g l u t a m a t e after kainate caused only a small further increase in fluorescence. The lack of additivity in the responses suggest that b o t h agonists depolarize cells by a similar m e c h a n i s m . C h a n g e s in intracellular free Ca 2+ as m e a s u r e d with fura-2 are shown in Fig. 2. The basal intracellular Ca 2+ was 217 +_21 n M (n = 23). A n increase in the K + concent r a t i o n from 5 to 25 m M resulted in a rapid increase in the intensity o f the fluorescence i n d i c a t i n g a rise in [Ca2+]i to a value of a b o u t 600 n M (Fig. 2A). This peak decayed within 1 m i n to a plateau. A similar transient response to K + has recently been observed in single cell recordings by using fura-2 in cerebellar g r a n u l e cells in explant cultures [2]. Since the increase in [Ca2+]i was n o t seen when 2 m M E G T A was added immediately prior to the a d d i t i o n of K + (data not shown), it most p r o b a bly represents a flow o f Ca 2+ into cells. The response to the a d d i t i o n of K ÷ was considerably reduced in the presence of verapamil (0.1 m M ) (Fig. 2B) a n d nifedipine (50 /tM) (Fig. 2C), blockers of voltage-gated Ca 2+ channels, i n d i c a t i n g that the rise in [Ca2+]i was due to Ca 2+ entry t h r o u g h v o l t a g e - d e p e n d e n t Ca 2+ c h a n n e l s opened by depolarization. K a i n a t e caused a sustained increase in the intracellular Ca 2+ concentration (Fig. 2D). Verapamil (0. I raM, in 6 experiments out of 6) (Fig. 2E) a n d nifedi-
60
A
Cai(nM)
B
Cai (riM)
Cai (riM) ~
fI ~
t K"
D
r290 L260
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Cai [riM)
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F
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t KA
t2 mint
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Cai{nM}
H "
~50
°
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U
230
..,..~f
180
NMDA
Fig. 2. Effects of verapamil and nifedipine on high-K +- and glutamate receptor agonists-induced changes in [Ca2+]i as measured with fura-2 in cultured cerebellar granule cells. The cells were loaded with fura-2 and recordings done as described in the text. In A, addition of K + (25 raM) was made as indicated by the arrow. In B and C, verapamil (0.l mM) and nifedipine (50 #M), respectively, were added 2 min prior to the addition of K + (25 mM). In D, application of KA (0.5 mM) was made as indicated, and in E and F, verapamil (0.1 mM) and nifedipine (50 #M), respectively, were added 2 min prior to KA (0.5 mM). In G, NMDA (0.25 mM) and quisqualate (QU, 0.25 raM) were added as indicated. In H, nifedipine (50 #M) was added 2 rain prior to NMDA (0.25 mM) and QU (0.25 raM). Original recordings are retraced.
pine (50 #M in 3 experiments out of 3) (Fig. 2F) had no significant effect on the kainate-induced rise in fura-2 fluorescence. Sequential additions of N M D A and quisqualate also caused increases in fluorescence (Fig. 2G), which were not significantly reduced in the presence of nifedipine (50 MM) (Fig. 2H). These results demonstrate that the mechanism of the rise in [Ca2+]i is different from that induced by high-K, and is not a result of depolarization-linked opening of voltage-gated Ca 2~ channels. The stepwise rise in cytosolic Ca 2+ by sequential additions of NMDA, quisqualate and kainate (Fig. 3A) was totally inhibited by a prior addition of EGTA (2 mM) (3B). Fig. 3C shows the dose response curves for the increase in fura-2 fluorescence by the agonists. Glutamate was the most effective in increasing cytosolic Ca 2+ with an EDs0 value around 5 #M in keeping with the data obtained in isolated hippocampal neurons [10]. The EDs0 values of the other agonists were 10 100 times higher than
61
A
100~
Cai (nM)
C
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-6 50
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I
1000
Fig. 3. Effects of N M D A , Q U and K A on [Ca-'+]i in cultured cerebellar granule cells as measured with fura-2. The recordings were carried out as in Fig. 2. In A, subsequent additions of N M D A ((I.5 mMt. QU (0.25 m M ) and KA (0.5 m M ) were made as indicated by the arrows. In B, E G T A (2.0 mM) was added immcdiately prior to N M D A (0.25 mM), Q U (0.25 m M ) and KA (0.25 mM). In C, the dose response curves lk~r G LU (O), KA (A), Q U (!~) and N M D A ( O ) are plotted as per cent of maximal fluorescence signal obtained by the addition o f i o n o m y c i n (1.0 ItM).
that of glutamate. N M D A had the smallest effect. The maximal response obtained with this agonist was only about 30% of that obtained with glutamate. The present results suggest that the glutamate receptor agonist-induced increase in intracellular Ca 9+ in granule cells is primarily due to Ca 2+ entry through Ca -~+permeable receptor coupled cation channels. The lack of an effect of glutamate receptor agonists in the absence of extracellular Ca 2+ as well as the insensitivity to verapamil and nifedipine are in agreement with this. Thus the increase in phosphatidylinositol turnover and gyanylate cyclase activation in response to glutamate [12, 13] might be the result of the increased Ca 2~ concentration as is the case with synaptoneurosomes [6]. Our results also show that cerebellar granule cells react to all three glutamate receptor agonists, kainate, quisqualate and N M D A with a rise in cytosolic Ca 2+ in contrast to spinal cord neurones [11], in which cells mainly the N M D A receptors seem to be linked to Ca 2+ influx. This study was aided by grants from the Academy of Finland, the Sigrid Jusdlius foundation and the Borg foundation. We thank Mr. Pekka Ketola for expert construction of equipment needed during this study. I Br~iuncr, T., Hfilster, D.F. and Strasser, R.J., Comparative measurements of membrane potentials with microelectrodes and voltage-sensitive dyes, Biochim. Biophys. Acta, 771 (1984) 208 216. 2 Connor, J.A., Tseng, H.-Y. and Hockberger, P.E., Depolarization- and transmitter-induced changes in intracellular Ca: + of rat cerebellar granule cells in explant cultures, J. Neurosci., 7 ( 19871 1384 1400. 3 Dupont, J.-L., Fournier, E., Gardette, R. and Crepel, F., Effect of excitatory amino acids on Purkinjc cell dendrites in cerebellar slices from normal and staggerer mice, Neuroscience, 12 (19841 613 6[9. 4 Grynkiewicz. G., Poenie, M. and Tsien, R.Y., A new generation of Ca 2+ indicators with greatly improved fluorescence properties, J. Biol. Chem., 260 (1985) 3440 3450.
62 5 Garthwaite, J. and Garthwaite, G., Cellular origins of cyclic GMP responses to excitatory amino acid receptor agonists in rat cerebellum in vitro, J. Neurochem., 48 (1987) 29 39. 6 Gusovsky, F., Hollingsworth, E.B. and Daly, J.W., Regulation of phosphatidylinositol turnover in brain synaptoneurosomes: stimulatory effects of agents that enhance influx of sodium ions. Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 3003 3007. 7 Hesketh, T.R., Smith, G.A., Moore, J.P., Taylor, M.V. and Metcalfe, J.C., Free cytoplasmic calcium concentration and the mitogenic stimulation of lymphocytes, J. Biol. Chem., 258 (1983) 4876-4882. 8 Holopainen, I. and Kontro, P., Glutamate release from cerebellar granule cells differentiating in culture: modulation of the K+-stimulated release by inhibitory amino acids, Neurochem. lnt., 12 (1988) 155 161. 9 Holopainen, I., Malminen, O, and Kontro, P., Sodium-dependent high-affinity uptake of taurine in cultured cerebellar granule cells and astrocytes, J. Neurosci. Res., 18 (1987) 479483. 10 Kudo, Y. and Ogura, A., Glutamate-induced increase in intracellular Ca 2+ concentration in isolated hippocampal neurones, Br. J. Pharmacol., 89 (1986) 191 -198. I I MacDermott, A.B., Mayer, M.L., Westbrook, G.L., Smith, S.J. and Barker, J.L., NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones, Nature (Lond.), 321 (1986) 519-522. 12 Nicoletti, F., Wroblewski, J.T., Novelli, A., Alho, H., Guidotti, A. and Costa, E,, The activation of inositol phospholipid metabolism as a signal transducing system for dicarboxylic excitatory amino acids in primary cultures of cerebellar granule cells, J. Neurosci., 6 (1986) 190:%1911. 13 Novelli, A., Nicoletti, F., Wroblewski, J.Y., Alho, H., Costa, E. and Guidotti, A., Excitatory amino acid receptors coupled with guanylate cyclase in primary cultures of cerebellar granule cells, J. Neurosci., 7 (1987) 40-47. 14 Sandoval, M.E. and Cotman, C.W., Evaluation of glutamate as a neurotransmitter of cerebellar parallel fibers, Neuroscience, 3 (1978) 199-206. 15 Tsien, R.Y., Pozzan, T. and Rink, T.J., Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator, J. Cell Biol., 94 (1982) 325 334. 16 Wroblewski, J.T., Nicoletti, F. and Costa, E., Different coupling of excitatory amino acid receptors with Ca 2 ~ channels in primary cultures of cerebellar granule cells, Neuropharmacology, 24 (l 985) 919 921.