the release and uptake of excitatory amino acids

David Nicholls and Dsvid Attwell

13~ this article, David Nicholls and David Attwell describe recent advances in our understamiing of the mechanisms by which excitatory amino acids are released from ceils, and of the way in which a low extracellular glutamate concentration is maintained. Glutamate can be released from cells by two mechanisms: either by CR’+-dependent vesicular release or, in pathological conditions, b?y reversal of the plasma membrane uptake carrier. The contrasting pharmacology and ionic. dependence of the glutamate uptake carriers in the vesicle membrane and in the plasma membrane explain how ghtamate (but probably not aspartate) can function as a neurotransmitfer, and why the extracellular glutamate concentration rises to neurotoxic levels in brain anoxia.

The majority of neurotransmitters are specifically synthesized com- pounds that are dedicated to their messenger role. It therefore came as a surprise in the early 1960s when electrophysiological studies showed that L-glutamate had a powerful excitatory action at a large number of synapses. In view of the ubiquity of glutamate, and the finding that L-aspartate could mimic many of its effects, it was initially difficult to accept that such a common metabolite could have a specific transmitter role. The problems with glutamate were exacerbated in the 197Os, when subcellular fractionation studies failed to reveal a significant amount of glutamate within a synaptic vesicle fraction that contained high concentrations of both acetylcholine and biogenic amines’. This led to hypotheses from a number of laboratories that amino acid neurotransmitters were released directly from the cytoplasm of nerve terminals, by mechanisms that differed funda- mentally from the exocytotic pathway for other transmitters.

In recent years these problems have been resolved with the demonstration of Ca2+-dependent vesicular release of glutamate, and the establishment of the compart- mentation of glutamate within the

nerve terminal (see Box 1). Two transport pathways are integral to the ability of glutamate to function as a neurotransmitter: first, a powerful uptake carrier located in both neurons and glia and capable of lowering the extracellular glutamate concentration to about 1 uM; and, secondly, a more specific transporter capable of packaging glutamate into a subpopulation of synaptic vesicles for subsequent exocytosis. In this article we re- view these developments.

Caz+-dependent release The release of glutamate from

synaptosomes - a model for the release of glutamate from intact synaptic terminals - may be monitored by including glutamate dehydrogenase and NADP+ in the synaptosomal incubation medium and following the increase in fluorescence produced as glutamate and NADP+ are converted to 2-oxoglutarate and fluorescent NADPH (see Fig. 1). Using this assay, it can be shown that 15% of the total glutamate content of a cerebral cortical or hippocampal synaptosome preparation can be released in a Ca2+-dependent manner by prolonged depolarization with KC1 (Ref. 2).

The Ca*+-dependent pool of glutamate in a synaptosomal preparation exchanges with added glutamate much more slowly than does the glutamate in the synaptosomal cytoplasm, consistent with release occurring from a non- cytoplasmic, presumably vesicular,

TiPS - November 2990 JVol. 22J

compartments. Release is highly dependent on the maintenance of high energy levels within the terminal, and any substantial decrease in the ATP/ADP ratio inhibits Ca2+-dependent release4s5, as is found for exocytosis in other systems.

Vesicular release of glutamate is expected to be triggered by a high localized cytoplasmic free Ca*+ concentration generated near active release zones by the Ca2f channels controlling exocytosis6. Ca*+ entering through these channels should be more efficiently coupled to glutamate exocytosis than is Ca+ entering nonspecifi- cally, for example catalysed by the Ca2+/2H+ ionophore iono- mycin. Thus, for a given average cytoplasmic [Ca2+] measured with fura-2, depolarization releases much more transmitter glutamate than does ionomycinr. The Ca*+- dependent release of glutamate by 30 mM KC1 is biphasicrs, with 20% exocytosed within 2 s and the remaining 80% released much more slowly with a tl12 of 70 s. This time course may be determined partly by the time course of the KCl-evoked Ca*+ entry into synaptosomes, which is also bi- phasic9, with a rapid phase (1 s) through inactivating channels followed by a slower phase (at least 10 s). However, the biphasic release of glutamate could also reflect an initial exocytosis of synaptic vesicles already docked at the exocytotic sites within the presynaptic active zones, followed by a slow phase occurring as the remaining synaptic vesicles are freed from cytoskeletal attachment and trans- located to the active zones.

Presynaptic regulation of glutamate exocytosis

On the basis of our own experiments with cerebral cortical synaptosomes (D. G. Nicholls, unpublished), there do not appear to be presynaptic NMDA- or AMPA- type glutamate autoreceptors on glutamatergic terminals. How- ever, kainate and glutamate can regulate glutamate release via a presynaptic receptor controlling chloride channelslo. Glutamate and quisqualate have also been shown to inhibit neuronal calcium currents via a G protein-linked mechanism”, and this action may provide negative feedback control of glutamate release. The gluta-

TiPS - Nouezzzber 2990 [Vol. 171 463

Pathways for the stora e, release and reu

pmynaptic

MlTOCHONDRlON \

extracellular

\

glia space

3Nd

K’ Glu-

Under resting conditions the glutamate concentrations in the extracellular space, the presynaptic cytoplasm and the lumen of glutamatergic synaptic vesicles are of the order of 1 PM, 10 mM and 100 mM, respectively. These gradients are maintained by two distinct electro- genie carriers. The acidic amino acid carriers present in the presynaptic (a) and glial (b) plasma membranes are Na+-dependent, show a high (Z-50 PM) affinity for L-glutamate, but also transport D- or L-aspartate’. The veside transporter (c) is Na+-independent, and accumulates glutamate anions driven by the internal positive membrane potential that is generated by a vesicular ATPase that pumps protons into the vesicle’ (d). This carrier has a low (mM) affinity for glutamate and

Glutamate may be reaccumulated directly into the nerve terminal or into adjacent glia. Giutamine svnthetase (e) is predominantly glial and some glutamine (Gin) can be formed from accumulated glutamate in this compartment. Glut- amine is present at about 0.5 mM in the extracellular space and can enter the

terminal by a low-affinity Na’-independent pathway (0. Glutamine may serve as a precursor for presynaptic cytoplasmic glutamate through the mitochondrial glutaminase (g). However, this enzyme is subject to potent product inhibition by glutamate itself and it is unclear what contribution this enzyme makes to the resynthesis of transmitter gJutamatea.

References

of transmitter ~~~tarnat~

does not transport aspartate. There is still controversy concerning the precise stoichiometries of these carriers.

Glutamate is normally released by Car+- dependent exocytosis; earlier ambi- guities concerning vesicular versus cytoplasmic release were due to difficulties in detecting the very labile storage of the amino acid within synaptic vesicless. However, the plasma membrane glutamate carriers may reverse their operation, releasing glutamate into the extracellular space, if the Na+ and/or K+ gradients are chronically decreased (see main text).

1 Erecinska, H. (1967) Biockm~. Dhnn~tncof. 36, 3547-3555 2 Maycox, I’. R., Hell, J. W. and jahn, R. (1990) Trenrls Neurosci.

13, 82-87 3 Nicholls, D. G. (1989) /. Ne~rroclrent. 52, 331-341 4 McMahon, H. T. and Nicholls, D. G. (1990) J. Nerrrochrm. 54,

373-380

mate analogue AP4 has been reported to inhibit the release of glutamate from hippocampal synaptosomes by acting at a presynaptic ~-Al’4 receptor12, but the molecular details of the inhibition, which is species specific, are unclear.

The best established presynaptic regulation of glutamate release is the inhibition produced by the GABAs agonist baclofen and by adenosine. This has been reported for both synaptosomesz3 and sliceszPz6 and is mediated by a pertussis toxin-sensitive G pro- iezn. More recently a muscarinic inhibition of glutamate release has been reported from hippocampal synaptosomest7.

Protein kinase C has been im- plicated in the control of release of a number of neurotransmitters, including glutamatets. Typically, a 30% increase of either basal or

KCl-evoked glutamate release from synaptosomes can be achieved by the addition of rather high concentrations of phorbol estersz9. This phorbol ester stimulation of glutamate release is greatly enhanced if transmitter release is evoked by 4-aminopyridine, which initiates spontaneous action potentials in the terminals (see Fig. 2)2n. The phorbol esters enhance both the 4- aminopyridine-evoked increase in cytoplasmic free [Ca*+] and the synaptosomal depolarization de- tected by cyanine dyes, suggesting that phorbol esters may prolong spontaneous action potentials, and increase the glutamate released per action potential. It is unclear what receptor system is responsible for activating protein kinase C in giu- tamatergic terminals izz viva.

Plasma membrane EAA carrier Glial cells and neurons possess

a similar plasma membrane glutamate uptake carrier2z which helps to terminate the postsynaptic action of neurotransmitter glutamate, and normally keeps the extracellular glutamate concentration below levels that damage neurons”. It is not known whether synaptically released glutamate is removed from the synaptic cleft mainly by uptake into the presynaptic neuron, or mainly by diffusion out of the cleft down the concentration gradient maintained by glial cell uptake. If radio- active glutamate is applied to the brain, most is taken up into glial cell@. However, although neurons may take up less glutamate than do glia, any uptake carriers in the presynaptic membrane will be ideally placed to remove glutamate after it has acted postsynap- tically. Glutamate removal appears to be much faster than the

464

I cytoplasmic Ca 2+ b glutamate release

340/380 nm excitation

505 nm emission

340 nm excitation

emission 460 nm

-1 0 400 800 1200 0 400 800 1200

time (s)

Fig. 1. P~ffeI fluffy assays of ~pt~~i cease free Ca2+ and gliitamate r&ease. a, b: Cytapfasmic free Ca**. f&a*+&, is dete.~in~in one aliquot of synaptosomes suspended in a fluorkmeter cuvette after oreloadmg with the ia;< indicator fora-2. In parallel, a second synaptosomal resuspension is incubated in medium containing, in addition, L-glutamate dehydrogenase (GDH) and NADP+. As glutamate is released it is oxidized to Poxoglutarate (200) by the enzyme and NADP+ is reduced to NADPH whcse fluorescence can be monitored. c, d: A typical experiment. Top tracas: rl-aminopyridine (4AP) elevates [Ca2+]Q by inhibiting presynaptic K+ channels responsiife for stab&ing the p&ma membrane ~tentiaf and a/~ng ~t~~ffs action potentials to develop? As a buffet gju~mate is &eased. Bottom Reces: in the preset of the Na + channet inhibitor tetrodotoxin mX) to i&bit the action potentials, the &AP-induced elevation in [Ca2+jc and the release of g/utamate are greatly reduced. However, the [Ca**], increase arld g/utamate release evoked by 30 mhi KCI still occur in the presence of mC. Glutamate release traces have been cm’rectti for basal release. (For details see Ref. 20.)

falling phase of the slow, NMDA receptor-mediated component of the postsynaptic current, the time course of which is determined by

_.

the properties of the NMDA receptor and associated ion channelz4. The plasma membrane glutamate uptake carrier is relatively non-

specific: it transports L- and D-

aspartate in addition to L-glutamate. Its apparent K,,, for binding extracellular glutamate is typically in the range 2-50 PM. (There may also be a low affinity glutamate uptake mechanism which, however, transports only a small fraction of the glutamate taken up at physiological glutamate concen-

Radiotracing trationsz5.)

studies have shown that the accumulation of glutamate into cells up its concentration gradient (--I VM outside, 10 mM inside cells) is driven by the movement of Na+ down its transmembrane electrochem- ical gradient. At least two Na+ ions are co-transported into the cell with each glutamate anion*s. There is also some evidence for the co-transport of protons into the cell on the uptake Carrie+.

Radiotracing experiments also suggested that intracellular K+ is necessary for glutamate uptake to operate*‘. However, since intra-

TiP5 - November 1990 Wof. 2 21

cellular K+ will generate a negative transmembrane potential, and thus increase the driving force for Na+ entry, it was unclear how much of the effect of K+ was a direct action on the uptake carrier and how much was an in- direct effect via a change of membrane potential. Recently, the whole-cell patch clamp technique has been used to resolve this problem (see Box 2). Removal of intracellular K+ or an increase of the extracellular K+ concen~a~on inhibits glutamate uptake by g&I cells even if the membrane potential is held constanP. Membrane depolarization also inhibits uptake. These data were explained by postulating that, for each glutamate anion transported into the cell, three Na+ {or two Na+ and one H+) ions are co-transported into the cell, and one K+ ion is transported out of the cell. With this stoichiornetry, and normal mammaiian ion concentrations, the carrier can theoretica~y maintain a glutamate concentration gradient of 280 000 : 1 (if three Naf are co-transported) or 23 000 : 1 (if two Na+ and one H+ are co- transported) across the plasma membrane.

Modulation of plasma men&ran%? glutamate uptake

When glutamate activates neuronal NMDA receptors, the resulting Ca2+ influx leads to activation of phospholipase AZ, and a release of arachidonic acid into the extracellular space29. Whole-cell clamp experiments have shown that even brief (2 min) exposure to l-10 PM

arachidonic acid produces a prolonged inhibition of glutamate uptake by glial cells39 This modulatory influence provides a possible means of interaction between neurons and glia. Its physiological significance is uncertain but, as discussed below, it may contribute to the failure of glutamate uptake that occurs during brain anoxia.

Source of transmitter glutamate Glutamate can be removed from

the synaptic cleft directly into the presynaptic neuron terminal or into glia, since the acidic amino acid carrier is present on both membranes (see above). Glial glutamate plays an integral role in the ‘glutamine cycle’, which has been proposed to regenerate terminal glutamate via glial gluta-

TiPS - November 1990 [Vol. 11 J

mine synthetase and diffusion of the glutamine so formed to the terminals for hydrolysis to glutamate (see Box 1). Such glutamate has been proposed to be incorpor- ated preferentially into the transmitter pooP1. While this cycle may well occur, some caution is necessary in the interpretation of experiments with brain preparations such as slices and synaptosomes, which may be contaminated with exposed mitochondria as a result of cell damage, since the glutaminase activity of such mitochondria will hydrolyse added glutamine to glutamate before uptake into the terminal*. What appears to be tne preferential release of a highly labelled pool of transmitter glutamate in the presence of added glut-mine may merely reflect this artifactual extracellular hydrolysis of the precursor. Indeed, since glutaminase experiences almost complete feedback inhibition in the presence of >20 PM glutamate*, it is difficult to see how glutaminase can function in the presence of the high cytoplasmic glutamate concentrations to be expected in the terminal. Further- more, even in intact terminals there is no obvious morphological explanation for glutamate gener- afecl in the cytoplasm by the mitochondria having priority over pre- existing cytoplasmic glutamate for incorporation into synaptic vesicles (see Box 1).

Synaptic vesicle glutamate transporter

Even though conventional synaptic vesicle preparations from brain regions believed to possess glutamate as a major transmitter contained negligible glutamate’, in the early 1980s it became clear that the preparations contained a subpopulation of vesicles which could accumulate exogen- ous glutamate in an ATP-dependent manneti2. Recently it has been shown that synaptic vesicles isolated in the presence of an ATP-regenerating system or N-ethylma!eimide (to prevent leakage from the synaptic vesicles) retain high concentrations of glutamate33. The vesicular glutamate transporterj4 can be readily distinguished from the plasma membrane acidic amino acid carrier since it is Na+-independent and displays a millimolar rather than micromolar affinity for gluta-

mate, consistent with the concentration of glutamate to be expected within the cytoplasm. The synaptic vesicle carrier is more specific than the plasma membrane caI-ier, transporting glutamate but not as- partate35. This discrimination between the amino acids at the level of the vesicle membrane throws into doubt the validity of employ- ing exogenously accumulated D-[3H]aspartate as a nonmetaboliz- able analogue of glutamate for release studies.

Aspartate as a neurotransmitter The Ca2+-dependent release of

endogenous L-aspartate from isolated terminal preparations is, at most, 10% of that seen for gluta- mate8, which would be consistent with the specificity of the synaptic vesicle carrier discussed above. In apparent contradiction to these results are those obtained with brain slices, where significant Ca2+-dependent release of aspar. tate is reported36. This apparent paradox may be due to the preparation of synaptosomes or synaptic vesicles selectively inactivating an aspartatergic subpopulation (although this would have to be true for all the alternative techniques that have been used). Alternatively the release of aspartate from slices

465

(that contain functioning synapses) may actually be nonvesicular if the operation of the plasma membrane amino acid carrier is reversed (see below) when KC1 or electrical stimulation is applied to depolarize the cells. More release by this route could occur when Ca2+ is present, since Ca2+ will allow vesicular release of excitatory transmitters (including glutamate), thus generating an extra depolarization and a Na+ influx that will raise the intracellular Na+ concentration - factors that promote the reversed operation of the uptake carrier. Which, if either, of these explanations is correct remains to be confirmed.

Caz+-independent release during anoxia/ischaemia

The extracellular K+ concentration in the brain, [K+],, rises excessively in pathological conditions such as an epileptic seizure, during ischaemia after a stroke, or during anoxia caused by perinatal asphyxia. A rise in [K+], will release more glutamate into the extracellular space in two ways. First, it will depolarize neurons, increasing their firing rate and increasing vesicular release of glutamate (if this is not already inhibited by a fall in ATP levels: see

Time(s)

Fig. 2. The protein kinase C activator 4t’Sphorbol dibutyrate (4B_PDBu) increases the glutamate release evoked by the repetitive action pofentials induced by I.aminopyridine (4AP) much more than it increases the release evoked by the ‘clamped’ depolarization produced by elevated KC/. In each trace the top curve represents the glutamare effiux in the presence of the phorbol ester, the centre curve that in the absence of the ester. while the bottom curve is the calculated difference between the two. Synaptoscmes were incubaled with glutamate dehydrogenase and NADP t as in Fig. 1. Where indicated, 1 !IM of 4/&P& or tire inactive isomer 4a-PDBu was added. h a the extensive stimubtion by 4fl-PDBu of 4AP-evoked glutamate release is seen: 4a-PDBu is comp/ete/y inactive (b), and on/y a small effect is seen upon KC/-evoked re;ease (c). Unpublished data of A. P. Barrie. T. S. Sihra and D. G. Nicholls.

TiPS - November 2990 jVo/. 2 21

e use of de-cell patch clam

The use of the whole-cell clamp technique to study the plasma membrane glutamate uptake carrier depends on

the fact that glutamate transport into the cell is electro- genie. The uptake of one glutamate anion is believed to be accompanied by the co-transport of three Na+ (or perhaps two Na+ and one Hf) ions into the cell, and by the counter-transport of one K+ ion out of the cell’, so that one elementary positive charge enters the cell for each glutamate taken up. As a result, glutamate uptake generates an inward membrane current, which can be recorded as the current flowing in the patch clamp circuit (a).

Whole-cell patch clamping offers three advantages. over studying glutamate uptake by radiotracing techniques. First, it allows control of the cell’s membrane potential. As shown in b this is a major determinant of the rate of uptake: depolarizing a glial cell from its normal resting potential of -80 mV to -18 mV greatly reduces the amount of uptake’l. Secondly, it provides good temporal resolution, allowing study of the kinetics of action of drugs that modulate uptake, such as arachidonic acidj. Thirdly, whole-cefl clamping provides good control of the composition of the solution

a

\ patch pipette

w

u glial csk

inside and outside the cell. This has revealed that (even with the cell membrane potential held constant) removal of intracellular K+ ions via the pipette almost abolishes uptake’, and that raising the extracellular K+ concentration inhibits uptake*c3 (c). As well as transporting glutamate into the cell (its normal direction of operation), the uptake carrier can also transport glutamate out of the cell when the extracellular K+ concentration is high and the cell is depolarized6 (d). The importance of the voltage and K+ dependence of upta!ce are discussed in the main text.

References 1 Barbour, 8.. Brew, H. and Attwell, D. (1988) Nat~rre 335,

433435 2 Brew, H. and Attwell, D. (1987) Nature 327,707-709 3 Saran&, M. and Athvell, D. (1990) Brain Res. 516,322-325 4 Wyllie, D. J. A., Mathie, A., Symonds, C. J. and Cull-Candy,

S. G. J. Physiol. (in press) 5 Barbour, B., Szatkowski, M.. Ingledew, N. and Attwell, D.

(1989) Natrlre 342, 918-920 6 Szatkowski, M., Barbour, 8. and A&well, D. Nature (in

press)

C d

2.5 rnM 1 57mM [K+],

u glutamate uptake - Glum

b Glu-

,oo p ~zl

PA

V -60 mV

glutamate uptake

glutamate release

Fig. a: Schematic diagram of a glial cell, with the glutamate uptake carrier in the plasma membrane, being whole-cell clamped by a patch pipette. Glutamate-evoked current through the pipette ref/ects activity of the uptake carder (provided there are no glutamate-gated ion channels in the cell membrane). b: Voltage dependence of uptake. inward currents generated in a whole-cell clamped salamander retinal g/is/ cell when glutamate uptake was activated by iontophoresed external g/utamate. The inward current and hence the amount of uptake is much smaller when ?he cell membrane potential, V,,,,,, is depoladzed to - 18 mV (as occurs during brain anoxia40) than at -80 mV. Adapted from Ref. 2. c: K+ dependence of uptake. The inward current generated by glutamate (30 /LM) uptake in a salamander retinalglial ce// (at -40 mV) is recuced when [K+!, is raised from 2.5 to 57 m,u, mimicking the [Kilo rise that occurs in brain anox/a@. Adapted from Ref. 1. d: Rdease of gkitamate by reversed uptake. Raising the K + concentration (from 0 lo 30 mM) around a salamander retinalglial cell, which is filled (via the pipette) with IO rnM Na l and 10 mMglutamate (i.e. physiologica! levels), generates an outward membrane current at 0 mV. This reflects the reversed operation of the uptake carder, with one K + entering the cell, and three Na + (or two Na + and one H +) and one g/utamate coming out of the cell. No glutamate was present in the external solution. From the study of Ref. 6.

TiPS - November 1990 /Vol. 111 467

uptake. The combination of these factors is sufficient to reduce the rate of uptake to less than 10% of its normal magnitudexn, severely compromising the mechanism that should prevent the glutamate concentration from rising to neurotoxic levels.

ATP + 1

F@. 3. Inhibition of glutamate uptake when [K ‘I0 rises in anoxia or ischaemia. On the left is shown the chain of events leading to a runaway increase in [K,], and [glutamate]O. On the right, shown as dashed fines, are processes inhibiting glutamate uptake and thus facifitating a rise in the extracellular gfutamate concentration to neurotoxic levels.

below); secondly, it will promote the release of glutamate (in regions where the extracellular glutamate concentration is low) by reversal of the plasma membrane glutamate uptake carrier (see below). The resulting rise in glutamate concentration will depolarize neurons further and thus release more K+ (see Fig. 3). This is a positive feedback system that tends to lead to a large rise in extracellular glutamate concentration. Unfortunately, however, if neurons are exposed to 100 PM glutamate for more than 5 min they diej7, probably as a result of an through exx;Fcha+s influx

22 (see

also Meldrum and Garthwaite, September TiPS).

Normally the plasma membrane glutamate uptake carrier prevents the extracellular glutamate concentration from reaching neurotoxic levels. However, as shown in

Fig. 3, several factors lead to a faiIure of glutamate uptake in the pathological conditions described above. First, a rise in [K’], inhibits uptake directly because it hinders the loss of counter-transported K+ from the carrier (see Box 2). Secondly, depolarization of the cell by the raised ]I(+], inhibits uptake (see Box 2j. Thirdly, arachidonic acid released by the

The extracellular glutamate concentration is, in fact, known to rise greatly during ischaemia or anoxia39. The origin of this extracellular glutamate is uncertain, however, because vesicular release of glutamate is inhibited when ATP levels fall after a few minutes anoxiaq,‘. On thermodynamic grounds the glutamate uptake carrier is expected to operate backwards, releasing glutamate into the extracellular space, when the transmembrane Na-, K+ and voltage gradients are greatly reduced in ischaemia or anoxia (if the extracellular glutamate concentration is lower than the vaIue at which there is no net glutamate transport). Patch clamp experiments have shown that this reversed uptake proceeds at a

high glutamate concentration in- reasonable rate (see Box 2). It hib,its uptakeJo. Finally, a decrease seems likely, iherefore, that much in INa+],,, which can result from of the glutamate released into the low ATP levels inhibiting the Na+ extracellular space during anoxia pump or from a large Na+ influx is Ca’+-independent, nonvesicular through glutamate- and voltage- release via reversed uptake. gated Na+ channels, also inhibits To support this idea, Fig. 4

-

Fig. 4. The minimum extracellular 300 - glutamate concentration that fheplas- ma membrane glutamate uptake carrier can maintain when the extracellular potassium concentration, [K ’ 1,. rises in anoxia or rschaemia, for two drffer- ent assumed stoichiometries of the uptake carrier. [Glutamate]0 was Cal- cufated by assuming that the carrier is operating at equilibrium. with no net glutamate transport across cell membranes. In other words, when [K’10 rises and depolarizes the cek, and [Na ‘I0 falls. the resulting decrease in thermodynamic ‘driving force’ forgluta- mate entry will result in the carrier running backwards and releasing glutamate into the extracellular space untif[glutamate], rises to reach a new

/ , ,

equilibrium value. [Glutamatel, was 0 10 20 30 40 50 60

calculated from the expressions26 for the carrier reversal potential, i.e.

IK(’ I, VW

V,,” =3 V,, -V, -V,,, if the carrier transports three Na and one g/u Into the ceffand one K ’ out of the cell. andV,, =2 V,,,, +V,-V,-V,,, if two Na , oneproton and onegtu are transported in and one K * is carried out. In these expressions V,,,,, VW V, and V, are the Nernst potentials for the ions, and V,,, is the membrane potential. To obtain igtutamate], as a Function solely of [K 11~. V, was assumed to equal Vk. V, was assumed to be zero. and[Na ‘I0 was taken as 143 mM-[K ‘]<, so that [Na * lo Falls by the same amount as fK ‘1, rises. For simp/ic;ty [Na ‘], and [K ‘4 n:‘ere assumed to remain constant at 25 mM and 115 mM; allowrng [Na ], to rise and [K ‘]! to Fall in anoxia and lschaemia would increase the calculated [glutamate],. The value of[g/utamatel, was set to 10 mM: any other choice of [g/utamate], would simp/y scale the value of [glutamate], acCOrdingtY.

shows the minimum extracelh.Iiar glutamate concentration that the uptake carrier can theoretically maintain when [K+], rises to different values (with associated cell depolarization and reduction of [Na+l,; see Fig. 4 legend for details of calculation). For a rise in [KC], to 60 mM, a corresponding cell depolarization to -20 mV, and a fall in [Natlo to 60 mM, as occur in anoxia40, reversed operation of the uptake carrier will raise the extrace!lular glutamate concentration until it reaches an equilibrium value of 75-250 PM (depending on the carrier stoichiometry assumed), i.e. sufficient to kill neurons3’.

References 1 DeBellerxhe, J. S. and Bradford, H. F.

(1977) /. Neamclrtva. 29, 335-.143 2 Nicholls, D. G. (1989) /. Nr~rroclrr~r. 52,

331-341 3 Wilkinson, R. and Nicholls, D. G. (1989)

ivelrrocheIr2. Brt. 15, 191-197 4 Kauppinen, R. A., McMahon, H. T. and

Nicholls, D. G. (1988) .Qztrosciertce 27, 175-182

5 Sanchez-Prieto, J. and Gonzalez, P. (1988) J. Neuroclrew. 50, 1322-1324

6 Smith, S. J. and Augustine. G. J. (1988) TreI& Nefrrosci. 11, 458-464

7 McMahon, H. T. and Nicholls, D. G. /. ~ele2rrochesI. (in press)

8 McMahon, H. T. and Nicholls. D. G. (1990) 1. Nertrocltent. 54, 37>Z80

9 Nachshen, D. A. (1985) /. Pkysiol. (Lott- don) 361, 251-268

10 Sarantis, M., Everett, K. and Attwe!l, D. (1988) Natzlre 332,451153

11 Lester, R. A. J. and Jahr, C. E. (1990) Netrron 4, 741-749

12 Gannon, R. L., Baty. L. T. and Terrian, D. M. (1989) Br& Res. 495, 151-155

13 Kate. K. and Fukuda, H. (1982) Gear. PhamIacor. 13. 445-44s

14 Potashner, S. j. (1979) J. Nel~rocArnr. 32, 103-1119

15 Burke, S. I’. and Nadler, J. V. (1988) I. Nertro</ran. 51, 1541-1551

16 Dolphin, A. C. and Preshvich, S. A. (1985) Nntlrrc 316, 148-150

17 Marchi, M., Bocchieri, I’., Garbarino, L. and Raiteri, M. (1989) Nenrosci. Left. 96, 229-234

18 Malenka, R. C., Madison, D. V. and Nicoll, R. A. (1986) Natwe 321, 175-177

19 Diar-Guerra, M. J. M., Sanchez-Prieto, J.. Bosca, L., Pocock, J. M., Barrie, A. P. and Nicholls, D. G. (1988) Biocki~v. Biophys. Acfa 970, 157-165

20 Tibbs, G. R.. Barrie, A. I’., Van- Mieghem, F., McMahon, H. T. and Nicholls, D. G. (1989) J. N~~rocltrm. 53, 169s1699

21 Kanner, B. 1. and Schuldiner, S. (1987) CRC Crit. Rev. Biockem. 22, l-38

22 Rothman, S. M. and Olney, J. W. (1987) Trends Nenrosci. 10, 299-302

23 McLennan. H. (1976) Bmbr Res. 115, 139-144

24 Lester, R. A. I., Clements, J. D., Westbrook, G. L. and Jahr, C. E. (1990) Nahrre 346, 565-567

25 Stallcup, W. B.. Bulloch, K. and Baetge, E. E. (1979) J. Neltroclrenl. 32, 57415

26 Erecinska, M., Wantorsky, D. an; Wilson, D. F. (1983) 1. Biol. Chern. 258, 9069-9077

27 Kanner. B. 1. and Sharon, 1. (1987) Biochert~isfry 17, 394%3953

2S Barbour, B., Brew, H. and Attwell, D. (1988) N&Ire 335, 433-435

29 Dumuis, A., Sebben, M., Haynes, L., Pin, J-P. and Bockaert, J. (1988) Nntlrre 336,68-70

30 Barbour, B., Szatkowski, M.. lngledew, N. and Attwell, D. (1989) Nattire 342, 918-920

31 Nicklas, W. J. and Krespan, B. (1982) in Nettrotrmsnritter hternctiou nrzd Com- pnrtaentntim (Bradford, H. F., ed.), pp. 383-395, Plenum Press

32 Naito, S. and Ueda, T. (1983) /. Viol. CJ~ef,z. 258, 696-699

33 Burger, P. M.. Mehl, E., Cameron, P. L., Maycox, P. R., Baumert, M., Lottspeich, F., De Camilli, I’. and Jahn, R. (1989) Neurorl 3, 715-720

34 Maycox, I’. R., Hell, J. W. and Jahn, R. (1990) Trends Neurosci. 13, 83-87

35 Naito, S. and Ueda, T. (1985) 1. Nezrro- clrertl. 44, 99-109

36 Szerh. J. C. (1988) !, Nerryochetv. 50, 219-224

37 Choi, D. W., Maulucci-Gedde, M. and

TiPS - November 1990 [Vol. 111

Kriegstein. A. R. (1987) J. Neltrochem. 7, 357-368

38 Attwell, D., Sarantis, M., Szatkowski, M., Barbour, B. and Brew, H. in Ex- citatory Amino Acids and Synaptic Fwtc- tion (Wheal, A. and Thomson, A., eds), Academic Press (in press)

39 Hagberg, H., Lehmann, A., Sandberg, M., Nystriim, B., Jacobson, 1. and Hamberger, A. (1988) J. Cereb. Blood Flow Metnb. 5,413-419

40 Siesjii, B. K. (1990) News Dhysiol. Sci. 5, 120-125

AP4: L-2-amino-4-phosphonobutanoate

o The TiPS series on the pharmacology of excitatory amino acids concludes next month with two features - one on the pharmacology of the metabotropic receptor by Daryle Schoepp, Joel Bockaert and Fritz Sladeczek, and the other on the molecular biology of non-NMDA ionotropic receptors by Eric Barnard and jeremy Henley.

Don’t live in England if you’re hypertensive Handbook of Experimental Pharmacology Volume 93: Pharmacology of Antihypertensive Therapeutics

edited by D. Ganten and P. J. Mulrow, Springer-Verlag, 1990. DM540.00 (xiv + 922 pages) ISBN 3 540 50427 3

This is an impressive book. De- spite having 55 authors and 31 chapters, it achieves a coherency, uniformity and completeness of coverage that is litt!e short of magisterial. After an opening chapter on treatment consider- ations, there are 11 chapters that cover the various classes of antihypertensive drugs, followed by chapters on interferences with 5-HT and dopamine. Next come eight ‘odd’ chapters on such varied topics as principles in the combination of antihypertensive drugs, electrolytes, stepwise treatment, the prognosis of hypertension, and toxicity testing. Following this are eight sections detailing traditions of antihyper-

tensive therapy in different regions of the world. The final chapter provides a comprehensive listing of antihypertensive medi- cation, giving doses forms and guidelines. However, there are no indications as to which countries the guidelines refer.

Depending on how the disease is defined, lO-20% of the world’s population has hypertension. Drug prescribing and drug use for the condition are cultural habits, not based solely on scientific principles. Furthermore, the relatively expensive, chronic treatment of symptomless condition is a luxury affordable by few, and of limited use in countries with life expect- ancies still in the low 40s. This perhaps explains why the world survey excludes Africa, the Arab nations, and the countries of South-East Asia. Antihyperten- sive therapy is only 50 years old. Over this period, it has changed from the treatment of small numbers of patients with very high blood pressure and end-stage dis- eases to the treatment of large numbers of patients with mild hypertension. Therapy has also

the release and uptake of excitatory amino acids

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