CaCI, (1.3 mM) and [3H]GABA were added at 5 min, and. [I4C]GABA at 55 min. At 59 min, 2.6 ~M-EGTA (sodium salt) was added to one experiment, followed by ...
617th MEETING, DUNDEE other details are those of actual proteins. For example, the amino acid compositions are taken from the sequences of real proteins given by Croft ( 1973). The user is required to nominate one of the proteins for purification. While the behaviour of all 20 proteins is modelled, only the enzyme activity of the nominated protein is revealed during simulated enzyme assays, the other 19 proteins providing the 'background' from which the nominated protein will hopefully be purified. Some information as to the temperature- and pH-stability of the nominated protein is given and then the user is offered the choice of separation techniques. These are modelled as accurately as possible, using the data on the proteins and data on separation media provided by the manufactures. For example, if ion-exchange chromatography is selected, the user is prompted to select either DEAE-cellulose or CM-cellulose and then to enter the pH value of the equilibration buffer. The charge on each protein in the mixture is then calculated using the pK values for amino acids in proteins given by Tanford (1961). The user is then asked whether a salt gradient or a pH gradient is to be used for elution and what its extent should be. The point on the gradient where each protein would elute is then calculated and a graphical display is given of the absorbance at 280 nm of the elution profile. The user is then given the opportunity to dilute the fractions and re-assay (maximum absorbance is initially set at 2.0, any higher absorbance will go off scale), to assay for enzyme activity. to run one or two-dimensional SDS polyacrylamide gels, to pool fractions, to repeat the step or to go home. Eventually, the user will hopefully locate and pool the fractions containing the enzyme activity, check the purity and apply another separation technique to the pooled material.
909 The program keeps a record of each purification step and costs each procedure. The aim is to purify each protein in as economic a way as possible. Material can be stored (recorded on disc) and retrieved later for further purification if necessary. Most importantly in simulation of this kind, the program will allow the user to make wrong decisions and then allow the experiment to fail as accurately as possible. For example, if too low or too high a pH value is selected, enzyme activity will be lost. The choice of the wrong medium or the wrong conditions of pH or salt concentration will produce results modelled as accurately as those resulting from the correct decisions. The program deliberately makes no attempt to describe the principles and practice of the separation techniques used. These are best taught by conventional methods. The user has to obtain the information on which to base his decisions in the same way that he would in the laboratory. i.e. go and ask someone or consult an appropriate publication. The program has proved very popular with students (and staff) and has been implemented on the IBM Personal Computer (with Color/Graphics adapter), the Apricot F series and the BBC model B Prior to formal publication. the program can be obtained at a small charge from the Biochemistry Microcomputer Group. at the above address. Croft. L. R. ( 1973) Hondhook of Prolein Seyuenci~s.Joynson-Bruvvers Ltd.. Oxford Tanford. C. (1961 ) Physicol Chemistry o/ Macromoli~cules.p. 556. John Wiley and Sons Inc. New York. London
Received 21 March 19x6
Ca2+-independent and Car+-dependent y-aminobutyrate release occur from distinct compartments within guinea-pig cerebral cortical synaptosomes TALVINDER S. SlHRA and DAVID G . NICHOLLS Neurosciences Research Group. Department of Psychiatry, Ninendls Medical School. University of Dundee, Dundw DDI 9 S Y . Scotland, U.K. Isolated synaptosomes contain a large pool of cytosolic y-aminobutyrate (GABA) which readily exchanges with external radiolabelled GABA via a plasma membrane carrier co-transporting the amino acid, N a + and probably CI (Kanner, 1983). A largely unresolved question is whether the amino acid released during physiological stimulussecretion coupling originates from this cytosolic pool, or from a largely speculative vesicular pool by analogy with other systems (Silinsky, 1982). In this paper two functionally distinct compartments of GABA are identified in isolated nerve terminals. One pool is labelled by exogenous [I4C]GABA within 5 min. is released in a Ca2+-independent manner by thermodynamic reversal of the GABA transporter (Blaustein & King, 1976; Sihra et al., 1984), and is consistent with the cytosolic pool. The second pool is only labelled by more prolonged exposure to exogenous GABA, but is released in a rapid, C a 2 + dependent manner consistent with a secretory mechanism involving the exocytosis of vesicular GABA. Synaptosomes were prepared from the cerebral cortices of Dunkin-Hartley strain guinea-pigs as described previously (Nicholls, 1978), and suspended (1.5 mg of protein/ ml) in 125 mM-NaCI, 3.5 mM-KCI, 0.4 mM-KH2P04, 5 mMAbbreviations used: GABA. y-arninobutyric acid. Tes. 2-{ [2-hydroxylI , I -bis(hydroxyrnethyl]arnino}ethanesulphonic acid.
Vol. 14
K
K GABA
\,
Time (s)
Time (s)
Fig. 1 . Diflkrmtial release. of ['HIGABA and ["CIGABA in the ahsence and presence of' external Ca2' Synaptosomes were incubated at a concentration of 1.5 mg of protein/ml. ['HIGABA ( I O n M , O.SpCi/ml) was added at 5 min and ['4C]GABA (0.45 PM, 0. I pCi/ml) at 55 min to the same incubation. Release of ['HIGABA and [I4C]GABAinduced by 30mM-KCI was monitored in the absence (0)or presence ( 0 ) of 1.3 mM-CaCI,.
910 NaHCO,, 20 m~ Tes (sodium salt), 1.2 mM-MgSO,, 10 mM-glucose and 1 mM-amino-oxyacetate, 30‘C, pH 7.4. CaCI, (1.3 mM) and [3H]GABA were added at 5 min, and [I4C]GABA at 55 min. At 59 min, 2.6 ~ M - E G T A(sodium salt) was added to one experiment, followed by 30 mM-KCI to both at 60min. The release of [I4C]GABA is independent of external Ca2+, whereas [3H]GABA release shows a marked C a 2 + dependency. In the presence of amino-oxyacetate. controls indicated no significant metabolism of the radiolabel, thus the difference in the release of the two isotopes indicates intrasynaptosomal compartmentation of GABA. From the topography of the nerve terminal, the relatively short incubation with [‘4C]GABAwould be expected to label the cytosol, while longer incubation of the same synaptosomal preparation with [3H]GABA evidently labels an additional compartment. From previous work in this and other laboratories (Blaustein & King, 1976; Kanner, 1983; Sihra et al., 1984) there is strong evidence that the CaZ+-independent release of [’HIGABA observed here occurs by the thermodynamic reversal of the GABA-transporter (Fig. I). Since a longer exposure to exogenous label is required before a C a 2 + dependent component becomes apparent, it is reasonable to
BIOCHEMICAL SOCIETY TRANSACTIONS assume that further translocation of the isotope into an internal compartment has to occur. By analogy with acetylcholine (Michaelson et al., 1978; Ceccarelli et al., 1980: Baba et al., 1983) and catecholamines (Douglas, 1968; Levi & Raiteri, 1978; Wedel et al., 1981), the results here suggests that the non-cytosolic compartment may be vesicular. Baba, A., Ohta. A. & Iwata, H. (1983) J. Ncwocham. 40. 1758 1761 Bbdustein. M . P. & King. C. A. (1976) J . M i h . B i d . 30, 153 173 Ceccarelli, B. & Hurlbut. W. P. (1980) Physiol. Rcw. 60,396441 Douglas. W. W. (1968) Br. J. Phurmcii,ol. C’humoihiv. 34. 451 474 Kanner. B. I. (1983) Biochcm. Biophys. Aciu 726. 293 316 Levi, G . & Raiteri, M. (1978) h i . Rev. Niurohiol. 19. 51 73 Michaelson, D. M.. Bilen. J. & Volsky. D. (1978) Bruin Rcs. 1-54, 409414 Nicholls, D. G. (1978) Biochem. J . 170. 51 I 522 Scott, 1. D. & Nicholls, D. G . (1980) Biochem. J. 186, 21 33 Sihrd. T. S.. Scott, I . G . & Nicholls. D. G. (1984) J. Ncuroc,hc~rn.43. I 6 2 4 I630 Silinsky, E. M . (1982) Fc.d. Proc. F i 4 A m . Soc. Eup. Biol. 41. 2169-21 7 I Wedel, R. J., Carlson. S. S. & Kelly, R. B. (1981) /‘roc,. N a i l . Actid. Sci. U.S.A.78. 1014 1018 Received 21 March 1986
N-Methyl-D-aspartate antagonists inhibit Ca2+ influx into hippocampal slices stimulated by depolarizing agents and by excitatory amino acids JANET M. CROWDER, MARTIN J. CROUCHER, H. F. BRADFORD and JAMES F. COLLINS Department of Biochemistr-v, Imperial College of‘ Science and Technology, London SW7 2 A Z , U . K .
Receptors for excitatory amino acids can be classified into three groups according to their selective activation by N methyl-r>-aspartic acid (NM DA), quisqualic acid and kainic acid (KA) (Watkins & Evans, 1981). Recently, excitatory amino acid receptor agonists have been shown to stimulate Ca2+influx into brain slices in vitro (Berdichevsky et ul., 1983; Lazarewicz et al., 1986). Moderate, brief, increases in intracellular Ca2+ mediate neurotransmitter release, whilst chronic high intracellular Ca’+ leads to excitotoxic cell damage (Farber, 1981). Thus, the ability of excitatory amino acid agonists to substantially increase cellular Ca2+content may play a role in brain disorders such as epilepsy, Huntington’s chorea and ischaemic brain damage. In the present study the effects of a homologous series of to-phosphonic-cr-carboxylic amino acids on basal and stimulated Ca’+ influx into hippocampal slices was investigated. Their actions on the stimulated release of neutrotransmitter amino acids were studied in parallel. The compounds tested were ( k )-2-amino-4-phosphonobutric acid (AP4), ( k)-2amino-5-phosphonovaleric acid (AP5) and ( f)-2-amino-7phosphonoheptanoic acid (AP7), of which the latter two are potent and selective antagonists of the NMDA-preferring receptor (Evans et ul., 1982). These compounds have previously been shown to have anticonvulsant properties (Coutinho-Netto et al., 1981; Croucher et al., 1982; Meldrum et ul., 1983). Hippocampal slices (300 pm thick) were prepared from SpragueeDawley rats and incubated in Tris-buffered Kreb’s medium containing 1.2 mM-CaC1, for 30 min before the
addition of 4sCaC12(Amersham International, 0.14 Ci/mol). The antagonists were added 5min before the addition of 45CaCI,and the depolarizing agents, veratrine (75 p ~or) K ’ ( 5 6 m ~ )were , added 1 min after the “CaCI,. Other methodology and analysis were as described by Bradford ot rrl. ( 1986). The drugs, AP4, AP5 and AP7, did not influence basal Ca’+ influx or basal amino acid efflux at concentrations up to 1 mM. However, at 1 @ 1 0 0 0 p ~all three antagonists partially ) veratrine inhibited Ca+ influx evoked by K + ( 5 6 m ~ or I ) . The maximum inhibition observed was ( 7 5 p ~ (Table ) 6&80%, compared with total inhibition after the addition of the general calcium antagonist (-)-D888, 1 0 ~These ~ . effects appeared to be receptor mediated as judged by the stereoselectivity of the actions of AP7. Thus, in accordance with both electrophysiological and behavioural studies (Croucher 6’1 a/., 1982; Perkins et al., 1982), the I>-( - ) isomer showed much greater activity than the L-( + ) (Table I ) . Whereas AP4 at 1 O p and ~ higher, in common with AP5 and AP7, partially inhibited glutamate and aspartate efflux evoked by veratrine and K + , the IC,,, concentration for AP4 was substantially higher than for the other two compounds (Table 1). None of the phosphono derivatives significantly ~
Table I . Inhibition of stimulated C k ’ uptake and c w > k i d glutamate release in hippocampal slices by e.ucitator.v amino acid antagonists ICs, values were graphically determined from log doscresponse curves. Abbreviation: Vt, veratrine.
AP4 (n = 6) Abbreviations used: NMDA N-methyl+-aspartic acid; KA, kainic AP5 (n = 6) acid; AP4, ( ~)-2-amino-4-phosphonobutyricacid; APS, ( f )-2-aminoAP7 (n = 6) 5-phosphonovdleric acid; AP7, (~)-2-amino-7-phosphonoheptanoic L-( +)-AP7 (n = 6) acid. U-( - )-AP7 (n = 6)
Ca’+ uptake (I~u PM) I. 220 19 32 > 2000 9
150 270 65 77
Glutamate release (ICw,W )
640 94 151
X70 207 144
13
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