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A Glutamate Receptor Regulates Ca2" Mobilization in Hippocampal Neurons (Excitatory Amino Acids/Calcium Stores) SHAWN N

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A Glutamate Receptor Regulates Ca2 Proc. Nat!. Acad. Sci. USA Vol. 85, pp. 8737-8741, November 1988 Neurobiology A glutamate receptor regulates Ca2" mobilization in hippocampal neurons (excitatory amino acids/calcium stores) SHAWN N. MURPHY AND RICHARD J. MILLER* Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, IL 60637 Communicated by Stephen J. Benkovic, August 15, 1988t ABSTRACT We investigated the effect of various excita- MATERIALS AND METHODS tory amino acids on intracellular free Ca24 concentration ([Ca24]i) in single mouse hippocampal neurons in vitro by using Embryonic mouse C57BL/6NHSD hippocampal neurons the Ca24-sensitive dye fura-2. In normal physiological solution, were cultured as monolayers as described (2) and used from glutamate, kainate, N-methyl-D-aspartate, and quisqualate all 3 to 14 days in culture. Cells were loaded with fura-2 in pentakis(acetoxymethyl ester) and checked for complete produced increases [Ca24]i. When all extracellular Ca2+ was deesterification with excitation spectra (2). The cells were removed, kainate and N-methyl-D-aspartate were completely bath-perfused and bath volume was exchanged every 7 sec. ineffective, but quisqualate and glutamate were able to produce Emissions from excitation at 340 nm and 380 nm alternating a spike-like Ca24 transient, presumably reflecting the release at 60 Hz were stored every second (17). Each wavelength was of Ca24 from intracellular stores. Ca24 transients of similar inspected for drug autofluorescence and changes in dye shape could also be produced by the a1-adrenergic agonist fluorescence. Fura-2 fluorescence typically decayed 10-20%o phenylephrine. After the production of a Ca24 transient a in 1 hr, which represented mostly dye leakage from cells second addition of quisqualate was ineffective unless intracel- when compared to nonirradiated controls. Estimation of lular stores were refilled by loading the cell with Ca24 following [Ca2e]i could be obtained by using calibration solutions (2) or depolarization in Ca24-containing medium. None of the con- by whole-cell calibration (8). Rmin, Rmax, and K were ob- ventional excitatory amino acid receptor antagonists inhibited tained by each method [0.367 ± 0.06, 14.5 ± 2.6, 3200 ± 160 the Ca24-mobilizing effects of quisqualate. Furthermore a- and 0.389 ± 0.03, 8.47 ± 1.3, 2360 ± 570, respectively (mean amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) + SD, n = 3)]. The former method allowed a more precise was unable to produce Ca24 mobilization in Ca24-free me- determination of the constants and was therefore used for dium, although it could produce Ca24 influx in Ca24- display of the data. All experiments were performed in containing medium. Thus, glutamate can produce mobilization Hepes-buffered Hanks' balanced salt solution (pH 7.45; of Ca24 from intracellular stores in hippocampal neurons by components, in mM: NaCl, 137; KCI, 5.4; MgSO4, 0.41; acting on a quisqualate-sensitive but AMPA-insensitive recep- MgCl2, 0.49; CaC12, 1.26; KH2PO4, 0.44; Na2HPO4, 0.64; tor. This receptor is therefore distinct from the quisqualate NaHCO3, 3; glucose, 5.5; Hepes, 20). Ca2+-free solutions receptor that produces cell depolarization. The possibility that were achieved by adding 20 ,uM EGTA to nominally Ca2+- this Ca24-mobilizing effect is mediated by inositol triphosphate free medium. Equimolar N-methyl-D-glucamine was substi- production is discussed. tuted for Na+ in Na4-free solutions. Quisqualate, a-amino- 3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and It is currently believed that the excitatory neurotransmitter ibotenate were purchased from Cambridge Research Bio- glutamate produces its effects by acting at at least three chemicals (Harston, U.K.) and found to be <1% glutamate separate types ofreceptors (1). This action leads to the gating by thin-layer chromatography. of a variety of ion channels that exhibit different permeabil- ities to monovalent cations and Ca2". An important conse- RESULTS quence ofthese actions is the influx ofCa2" into neurons and an increase in the intracellular concentration of free Ca2" Experiments were performed with single mouse hippocampal (2, 3). This increase is thought to be essential neurons in vitro (n = 84). [Ca2`]j was measured by micro- [Ca2+]i for fluorimetry using the Ca2+-sensitive dye fura-2 (17). We promoting glutamate-induced changes in neuronal activity (4) examined the effects of excitatory amino acids with the and synaptic plasticity (5) as well as cell death in pathological specific intention ofdetermining whether they could mobilize situations (6). Previous work in our own and other laborato- Ca2+ from intracellular stores. As we have previously dem- ries has demonstrated that glutamate stimulates Ca24 entry onstrated that activation of excitatory amino acid receptors into neurons through both N-methyl-D-aspartate (N-Me-D- produces Ca2+ influx due to the activation ofion channels (2), Asp)-gated ion channels and voltage-sensitive Ca2+ channels it was first necessary to establish conditions under which (2, 3). We now report that glutamate can also increase [Ca2+]i such effects were not observed. Fig. 1 demonstrates the by mobilizing intracellular Ca2" stores. This effect is inde- pendent of the presence of external Ca2` and is mediated by Abbreviations: AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazole- a glutamate receptor that may not be linked to an ion channel propionate; [Ca2+]i, intracellular concentration of free Ca2+; N-Me- and whose specificity appears distinct from those of other D-Asp, N-methyl-D-aspartate; InsP3, inositol trisphosphate. glutamate receptors. *To whom reprint requests should be addressed at: Department of Pharmacological and Physiological Sciences, University of Chi- cago, 947 East 58th Street, Chicago, IL 60637. The publication costs of this article were defrayed in part by page charge tCommunication of this paper was initiated by Emil Thomas Kaiser payment. This article must therefore be hereby marked "advertisement" and, after his death (July 18, 1988), completed by Stephen J. in accordance with 18 U.S.C. §1734 solely to indicate this fact. Benkovic. 8737 Downloaded by guest on September 25, 2021 8738 Neurobiology: Murphy and Miller Proc. Natl. Acad. Sci. USA 85 (1988) 1200 r 900 F CM 600 F Cu 0 300 F -j QL 50 mM K+ 1 0 gm Q 300 M KA - Ca+ 0 10 20 30 40 Time, min FIG. 1. Quisqualate (Q) but not kainate (KA) increases [Ca2+], in Ca2l-free medium. In this hippocampal neuron, depolarization with 50 mM K+ produced a large increase in [Ca2+],. In Ca2+-free medium (-Ca2+), kainate was ineffective but quisqualate produced a Ca2+ transient. When the neuron was returned to normal medium, kainate produced a large increase in [Ca2+],. On switching back to Ca2+-free medium, [Ca2+], fell and then rose again when Ca2' was reintroduced. effects of kainate in both normal and Ca2"-free media. In pleted in a time-dependent manner when the external Ca2" Ca2"-free medium kainate produced no effect. However, in concentration is reduced. Because of this lability, we always normal medium kainate produced a large increase in [Ca2+]i challenged cells with agonist in Ca2"-free medium 3 min after similar in form to increases in [Ca2"]J produced by depolar- Ca2" loading (except under special circumstances; see Fig. 2 ization with 50 mM K+. When the external Ca2" was A and B). Application of quisqualate to glial cells never removed during the kainate application, [Ca2+] immediately produced an increase in [Ca21]i. In neurons, Ca2+ transients fell back toward basal levels only to increase once more on could be produced in Ca2+-free medium by glutamate (Fig. the reintroduction of Ca2". In Ca2--free medium, depolar- 2C) (EC50 = 30 ,uM) and by ibotenate (EC50 = 100 ,uM) but ization of cells with 50 mM K+ or addition of N-Me-D-Asp not by kainate (Fig. 1) or N-Me-D-Asp (Fig. 2D) even at (Fig. 2) also produced no increase in [Ca2+]i. These results extremely high concentrations. Furthermore, coapplication indicate that under the Ca2+ free conditions used in these of either kainate or N-Me-D-Asp (Fig. 2D) did not reduce the studies, activation of Ca21 influx is not observed. effects of quisqualate. Quisqualate proved to be a potent In contrast to the above observations, Figs. 1 and 2A stimulus (EC50 = 200 nM). No response was normally illustrate the effect of quisqualate on [Ca2+]i in Ca2`-free observed at 10 nM quisqualate, but clear responses were seen medium. Quisqualate was equally effective in the presence of at 100 nM, with maximal effects at 10 ,uM (Fig. 2E). The tetrodotoxin (1 AM) or when all external Na+ had been absolute magnitude ofthe effect produced by quisqualate was replaced by N-methyl-D-glucamine. Quisqualate produced a quite variable, however: A[Ca2+]i = 163 ± 26 nM (mean ± clear spike-like Ca2+ transient that, under these conditions, SEM, n = 29). An interesting feature of the effect produced presumably reflects Ca2+ release from an intracellular store. by quisqualate is that it showed only minor desensitization. A second challenge with quisqualate produced no detectable In Fig. 2F we compare the effect of multiple additions of effect, indicating that the stores had become depleted after quisqualate to similar challenges with the a1-adrenergic the initial challenge (Fig 2A). However, we found that agonist phenylephrine. Multiple responses to quisqualate quisqualate-sensitive stores could be rapidly refilled if we could be obtained in a single cell under the appropriate loaded cells with Ca2+ by depolarizing them with 50 mM K+ conditions, but the response to phenylephrine in some cells in Ca2+-containing medium. This allows Ca2+ entry through was rapidly desensitized in spite of attempts to refill phenyl- voltage-sensitive Ca2+ channels (2). On switching back to ephrine-sensitive stores by the same procedure that proved Ca2+-free medium a further quisqualate-induced transient successful with quisqualate.
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