Journal of Neurochemistry
Raven Press, Ltd., New York 0 1991 International Society for Neurochemistry
Excitatory Amino Acid Receptors Coupled to the Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum During Development E. Southam, S. J. East, and J. Garthwaite Department of Physiology,University of Liverpool, Liverpool, England
preparations by measuring cyclic GMP accumulation. In the immature tissue, N-methyl-D-aspartate(NMDA) and glutamate were highly efficacious agonists, whereas a-amino-3hydroxy-5-methyl-4-isoxazolepropionate(AMPA) and quisqualate evoked only small responses. The effect of glutamate at all concentrations tested (up to 10 mM) was abolished by the NMDA antagonist, (+)-5-methyl-l0,1I-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-iminemaleate (MK-80I). In adult slices, AMPA and quisqualate were much more effective and their effects were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist for ionotropic non-NMDA receptors, whereas the apparent efficacy of NMDA was greatly reduced. The major changes took place between 8 and 14 days postnatum and, in the case of NMDA, part of the loss of sensitivity appeared to reflect a decline in the ambient levels of glycine with age. Moreover, a component of the response to glutamate in the adult was resistant to MK-80 I .
Cyclic GMP accumulations induced by NMDA and nonNMDA agonists alike were Ca’+-dependent and could be antagonized by competitive NO synthase inhibitors in an arginine-sensitive manner, indicating that they are all mediated by NO formation. With one of the inhibitors, L - N ~ nitroarginine, a highly potent component (ICso= 6 M)evident in slices from rats of up to 8 days old was lost during maturation, indicating that there may be a NO synthase isoform which is prominent only in the immature tissue. Cyclic GMP levels in adult slices under “basal” conditions were reduced markedly by blocking NMDA receptors, by inhibiting action potentials with tetrodotoxin, or by NO synthase inhibition, suggesting that the endogenous transmitter released during spontaneous synaptic activity acts mainly through NMDA receptors to trigger NO formation. Key Words: Glutamate-N-Methyl-D-aspartate receptors-Nitric oxide-Cyclic GMP-Cerebellum-Development. Southam E. et al. Excitatory amino acid receptors coupled to the nitric oxide/cyclic GMP pathway in rat cerebellum during development. J. Neurochem. 56,2072-208 I (1 99 1).
Studies on slices and dispersed cells from the cerebellum of immature rats have shown that activation of excitatory amino acid (EAA) receptors triggers the Ca2+-dependent synthesis of the novel intercellular messenger, nitric oxide (NO), from L-arginine (Garthwaite et al., 1988, 1 9 8 9 ~ )A . major action of the NO so formed is to stimulate the enzyme guanylate cyclase and thus trigger cyclic G M P (cGMP) synthesis. In the immature tissue, this effect of glutamate appears to be mediated mostly, if not entirely, through the N-methylD-aspartate (NMDA) class of receptor (Garthwaite, I985), even though the non-NMDA receptor agonist, kainate, is also able to evoke, through NO, a large ac-
cumulation of cGMP (Garthwaite et al., 1989b). The other selective non-NMDA receptor agonists, quisqualate and c~-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), even at high concentration, induce only small cGMP elevations. The question inevitably arises as to the relevance of these findings to the adult. During the postnatal development of the cerebellum, changes take place in the relative depolarizing potency and efficacy of selective glutamate receptor agonists. In particular, there is an apparent loss of chemosensitivity to NMDA of both principal neuronal types, i.e., granule cells and Purkinje cells (Dupont et al., 1987; Garthwaite et al., 1987).
Received September 24, 1990; revised manuscript received November 25,1990; accepted December 5, 1990. Address correspondence and reprint requests to Dr. J . Garthwaite at Department of Physiology, University of Liverpool, Brownlow Hill, P.O. Box 147, Liverpool L69 3BX, U.K. A bbreviarinns used: AMPA, ol-amino-3-hydroxy-5-methyl-4-isox-
azolepropionate; AP5, D-2-amino-5-phosphonopentanoate; cGMP, EAA, cyclic GMP; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; excitatory amino acid; MK-80 1, (+)-5-methyl-l0,1l-dihydro-5H-dibenzo[a,d]cyclohepten-5,I0-iminemaleate; N-NARC, L-NG-nitroarginine; NMDA, N-methyl-D-aspartate; L-NMMA, L-p-monomethyl-L-arginine; NO, nitric oxide; TTX, tetrodotoxin.
Abstract: The coupling of excitatory amino acid receptors to the formation of nitric oxide (NO) from arginine during the postnatal development of rat cerebellum was assayed in slice
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homogenized by sonication. The protein content of the homogenate was determined by the automated Lowry method and, following centrifugation, the cGMP content of the supernatant was measured by radioimmunoassay (see Garthwaite and Garthwaite, 1987). Results are expressed as mean cGMP levels per milligram of protein (or percentage of control levels) -t SEM, and statistical comparisons were made using the Student's t test; n values refer to the number of slices used and, within any experimental group, each slice was from a different animal. In experiments requiring a Caz+-freemedium, slices were transferred, 15 min before the addition of test compounds, to a solution in which CaC12 had been replaced by 1 mM EGTA. The same preincubation period was adopted when antagonists acting at the receptors or on the NO synthase were tested.
Nevertheless, in the adult cerebellum in vivo, activation of afferent excitatory pathways is known to give rise to large elevations in cerebellar cGMP levels (Biggio and Guidotti, 1976; Rubin and Ferrendelli, 1977; Lundberg et al., 1979; Wood et al., 1982). Moreover, the NOsynthesizing enzyme, NO synthase, is known to be present in adult rat brain (Knowles et al., 1989; Bredt and Snyder, 1990; Forstermann et al., 1990). Accordingly, the aim of the present study was to investigate the coupling of EAA receptors to the NO/ cGMP system in adult rat cerebellum and, in the light of differences observed in comparison with the immature tissue, to chart the changes that occur during the maturation of this brain region.
Materials
MATERIALS AND METHODS
AMPA, D-2-amino-5-phosphonopentanoic acid (APS), and 6-cyano-7-nitroquinoxaline-2,3-dione(CNQX) were from Tocris Neuramin; kainic acid, NMDA, quisqualic acid, LNG-nitroarginine(N-NARG), sodium nitroprusside, and tetrodotoxin (TTX) were from Sigma; (+)-5-methyl-10,l l-dihydro-5H-dibenzo[a,d]cyclohepten-5,lO-iminemaleate (MK-801) was from Merck, Sharpe and Dohme; and L-NGmonomethyl-L-arginine (L-NMMA) was supplied by the Wellcome Research Laboratories.
Methods Rats (Wistar origin, either sex) were killed by decapitation. For animals aged 21 days and over, the cerebellum was excised and the hemispheres removed by parasagittal razor cuts. One of the cut surfaces of the vermis was glued (with cyanoacrylate) onto the stage of a Vibroslice (Campden Instruments) and then embedded in 3% agar (made up in 0.9% saline and kept stirred at 37°C). The agar formed an exoskeleton which greatly enhanced the stability of the tissue during sectioning. Slices were cut at 400-pm intervals in cool ( 1 2- 14°C) Krebs-Henseleit solution containing (in mM) NaCl ( 120), KCI (2),CaC12(2),MgS04( I . 19),KH2P04(1.1 8), NaHC03 (26), and glucose ( I l), gassed with 5% C02in 02. pH 7.4. Subsequently, the slices were allowed to recover for about 30 min in gassed solution maintained at 37°C in a shakingwater bath. The slices were then redistributed so that each incubation vessel contained slices (usually four) from different animals, and the preincubation was continued for about an additional hour. For younger animals, slices of vermis were cut in the parasagittal plane using a tissue chopper (see Garthwaite and Garthwaite, 1987), but the procedures otherwise were the same. At predetermined times after the addition of test compounds, slices were quickly removed from the incubating medium and inactivated for 3 min in 200 pI of boiling 50 mMTris, 4 mMEDTA buffer, pH 7.6. The slices were then 90
la)
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cGMP responses to EAA receptor agonists The mean cGMP level in unstimulated adult (25week-old) cerebellar slices was 1.75 k 0.13 pmol/mg of protein (n = 85). Addition of the non-NMDA agonists, kainate, AMPA, and quisqualate, evoked a prompt rise in c G M P levels which peaked within 30 s, whereas the response to NMDA was slower (Fig. la). Subsequent experiments used a 2-min exposure for NMDA and 30-s exposures for the others. Concentration-response curves (Fig. 1b) showed that the apparent efficacy of kainate was higher than that of AMPA, the responses at or near their maximally effective concentrations (100 p M ) being about 80 and
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FIG. 1. Time courses (a) and concentration-response curves (b)for cGMP levels in adult rat cerebellar slices exposed to kainate (O), AMPA (0),quisqualate (O), and NMOA (m), each at a concentration of 100 pA-4 (n = 3-4).
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50 pmol/mg of protein, respectively. The corresponding ECSo values were approximately 20 and 10 pM. The effects of NMDA and quisqualate were also concentration-dependent, but no clear maxima were obtained: at the highest concentrations tested ( 100 p M quisqualate, 1 M N M D A ) , cGMP levels were similar to those observed with 100 pM AMPA. Developmental changes. In terms of absolute cGMP levels, the response to kainate, at a concentration (100 p M ) which is maximally effective in adult and immature tissues (Garthwaite, 1982),remained relatively constant throughout development (see legend to Fig. 2), and so this was used as the reference. In agreement with previous findings(Garthwaite, 1982),NMDA (100 p M ) evoked a very large accumulation of cGMP in slices from 8-day-old animals (Fig. 2), the levels attained being about twice those produced by kainate. However, a striking reduction occurred during maturation, particularly between 8 and 14 days, so that in the adult, the response to 100 pM NMDA was only 10%of that to kainate. In contrast, the effect of AMPA (100 p M ) became larger during development (20% of the response to kainate at 8 days rising to 70% in the adult), the major change again taking place between 8 and 14 days. Quisqualate (100 p M ) , though starting from a lower base, behaved similarly to AMPA (Fig. 2). One factor that could contribute to the loss of response to NMDA is a decline in the ambient levels of glycine. Glycine binds with high affinity to a site on the NMDA receptor, and occupation of this site is necessary for opening of the NMDA receptor channel (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988);glycine also may inhibit a component of NMDA receptor desensitization (Mayer et al., 1989). DSerine 250
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Age (days) FIG. 2. Developmental changes in cGMP responses to kainate
(0, AMPA (0), quisqualate (O),and NMDA (m), all at 100 pM. The data are expressed as percent mean response to 100 pM kainate at each age; the absolute values for these reference responses were 87 k 6, 114 & 4, 71 k 5, and 70 f 3 pmol/mg of protein at 8, 14, 21, and >30 days, respectively (n = 4-8).
J. Neurochem., Val. S6. No. 6, 1991
is an agonist for this glycine site but, unlike glycine itself, is not transported into cells nor does it activate inhibitory glycine receptors. The dose-response curve for NMDA was not affected significantly by D-serine in slices from 8-day-old animals but, in the adult, cGMP accumulations at 100 and 300 pMNMDA were significantlylarger (Fig. 3). Developmentally, this augmentation by D-serine became evident at 21 days of age (Fig. 4). Effect oJCNQX. CNQX is generally regarded as a fairly selective non-NMDA antagonist, but because of its activity on the glycine site of the NMDA receptor, it can also inhibit NMDA responses when glycine levels are low (Watkins et al., 1990).In the immature (8-dayold) cerebellum, CNQX inhibits the cGMP response to kainate, but not that to NMDA (Garthwaite et al., 19896). In slices of the adult, however, CNQX inhibited not only responses to kainate, AMPA, and quisqualate (each at 30 p M ) , but also that to NMDA (300 pM), the ICsOvalues (2-4 p M ) being similar for all agonists (Fig. 5). The inhibition by CNQX of the NMDA response, but not of the response to AMPA, could be nullified, however, by including D-Serine in the incubation medium (Fig. 6). Involvement of NO Addition of L-arginine, the substrate for the NO synthase enzyme, increased the cGMP response to AMPA in adult cerebellar slices in a dose-dependent manner (Fig. 7). The effects were maximal (50% increase) at 100-300 pM arginine and then declined. The analogue, L-NMMA (100 p M ) , which is a competitive inhibitor of the formation of NO from arginine (Knowles et al., 1990), markedly reduced the responses to AMPA, quisqualate, kainate, and NMDA (Fig. 8). The ICsOvalue for L-NMMA against AMPA was about 6 pM(data not shown), and its inhibitory effects against all agonists could be counteracted by additional arginine (Fig. 8). Another NO synthase inhibitor, N-NARG, is a more potent inhibitor than L-NMMA of the NMDA-induced cGMP formation in slices of immature cerebellum, where it exhibits a strongly biphasic inhibition curve (ICso = 6 and 600 nM,Fig. 9a; East and Garthwaite, 1990). In the adult tissue, by contrast, the curve was a simple monophasic one (ICs0= 600 nM; Fig. 9a). During development, the loss of the higher potency component was virtually complete by 15 days (Fig. 9b). The cGMP elevations produced by NMDA and nonNMDA receptor agonists alike were Ca2+-dependent: preincubating adult slices in Ca2+-freemedium for 15 min reduced the subsequent response to 300 pM NMDA from 28 k 3 to 2.1 4 0.6, the response to 100 pM AMPA from 45 2 2 to 1.4 f 0.3, and the response to 30 pMquisqualate from 24 f 2 to 2.2 +- 0.5 pmol/ mg of protein (all p < 0.00 1, n = 4). A compound which releases NO spontaneously, sodium nitroprusside (100 pM, 5-min exposure) raised cGMP levels in adult slices to 65 f 3 pmol/mg of pro-
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lCNQXl [pM) FIG. 5. Inhibitionby CNQX of the cGMP elevations induced by 30 FIG. 3. Concentration-cGMP response relationships for NMDA in cerebellar slices from 8-day-old (circles) and adult (squares) rats in the absence (filled symbols) and presence (open symbols) of 100 f l D-serine. Responses were significantly increased by the amino acid only at 100 jtM and 300 NMDA in the adult ( p < 0.05 and 0.001, respectively; n = 4-12).
tein (n = 8). In immature (8-day-old) slices, the corresponding increase was to 105 t 12 pmol/mg of protein (n = 4). Maintenance of basal cGMP levels and effect of n x In slices of the adult, unstimulated levels of cGMP were reduced by either L-NMMA (100 F M ) or TTX (1 p M ) to 20-30% of controls. Also, CNQX and the NMDA antagonist, AP5, each lowered the levels by 50%, but the inhibition by CNQX could be overcome by adding D-serine (Fig. 10).
jtM concentrationsof kainate(O), AMPA (0),and quisqualate (O), and by 300 pVl NMDA (m) in adult cerebellar slices (n = 3-4).
We also tested whether the increases in cGMP induced by receptor agonists were sensitive to TTX in the adult tissue (Fig. 11). This proved not to be the case for AMPA and kainate (and also glutamate); however, with NMDA, cGMP levels were reduced by about 40%. No such inhibition by TTX was observed in the presence of D-serine.
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FIG. 4. Developmentalchanges in the enhancing effect of D-serine (100 pM) on cGMP responses to 300 jtM NMDA (*p < 0.002; '*p < 0.001; n = 4-12).
FIG. 6. In adult slices, o-serine (100 p.M) does not affect the inhibition by CNQX (10 f l )of the cGMP response to 30 jtM AMPA (a), but counteracts that to 300 f l NMDA (b). In the absence of D-serine, CNQX significantly inhibited responses to both NMDA (p < 0.01)and AMPA (p < 0.001). o-Serinealone selectively increased the response to NMDA ( p < 0.001). With o-serine present, the degree of inhibition by CNQX of the response to AMPA was similar ( p < 0.001), whereas that to NMDA was nullified (n = 4).
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FIG. 7. L-Arginine augments cGMP response to AMPA (100 &f)
in adult cerebellar slices in a dosedependent manner. Significant changes occurred at arginine concentrationsof 30 pM and above ( p < 0.05-0.001; n = 6-8).
Actions of glutamate Glutamate increased cGMP levels in slices of both the 8-day-old and adult in a dose-dependent manner (Fig. 12). Because of the presence of uptake mechanisms, it is necessary to use high concentrations of glutamate for it to penetrate the tissue extracellular spaces; this, in turn, makes it very difficult to study the pharmacology of its effects with competitive antagonists like CNQX and AP5, because correspondingly high antagonist concentrations are needed and then their selectivity becomes questionable (Garthwaite, 1985). Ac-
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FIG. 8. L-NMMA (100 pA4) inhibits cGMP responses to 100 pM
AMPA, quisqualate, kainate, and NMDA, and this inhibitioncan be neutralized by additionalarginine, becominginsignificantwith 300 phd arginine (see key; n = 4).
J. Neuroehem., Vol. 56, No. 6, 1991
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FIG. 9. a: Inhibition of cGMP responses to 300 pM NMDA by N-
NARG in adult (m)and 8-day-old (0)slices showing, only in the latter, a component sensitive to low nanomolar concentrationsof N-NARG. Values for the control responses were 35 ? 4 (adult, n = 7) and 161 f 19 (8 days, n = 11) pmol of cGMP/mg of protein. For the other data, n = 3-1 7. Displayed in b are the developmental changes in the inhibitory effects of 100 nM and 3 pM N-NARG, showing loss of the high potency component by 15 days of age (note logarithmic scale). The control responses to 300 pM NMDA were as follows (in pmol of cGMP/mg of protein): 46 f 6 (3 days), 115 k 7 (5days), 161 5 19 (8 days), 89 k 9 (11 days), 78 k 6 (15 days), and 35 k 4 (29-33 days) (n = 4-12).
cordingly, we used the noncompetitive NMDA antagonist, MK-801, to determine the extent to which NMDA receptors contribute to glutamate-induced cGMP formation. In 8-day-old cerebellar slices, the effects of glutamate at all concentrations tested were abolished by MK-80 1 at a dose (10 p M ) which completely inhibited the cGMP response to NMDA (Fig. 12a). In the adult slices, however, a component remained in the presence of MK-801 (Fig. 12b). This MK-80 1-resistant portion of the response became larger as the glutamate concentration was raised (36% at 1 mM, increasing to 68% at 10 mM). DISCUSSION Because of the experimental advantages offered, most of the previous work dealing with the coupling of EAA receptors to NO synthesis has been camed out
EXCITATORY AMINO ACIDS AND NITRIC OXIDE
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“basal” cGMP levels in adult cerebellar slices. The first column (a) corresponds to levels in control slices (set at 100%; absolute value = 2.94 f 0.16 pmol of cGMP/mg of protein; n = 14). In the other columns, the slices were incubated with the following: b, CNQX (10 pM, n = 4); c, AP5 (50pM, n = 8); d, CNQX plus AP5 (n = 4); e, L-NMMA (100 f l ,n = 8); f, TTX (1 pM, n = 8); g, o-serine (100 pM, n = 4); h, CNQX plus o-serine (n = 4). *p < 0.05,**p < 0.01 compared to controls.
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on preparations of the immature cerebellum, and the question inevitably arose as to the relevance of the findings to the adult. The present study finds some basic similarities, but also some differences that could have a significant bearing on our present understanding of the physiological regulation of this novel messenger system. NO as the mediator of cGMP responses The first conclusion to be made is that the cGMP elevations taking place in response to EAA receptor
AMPA Kainate NMDA NMDA
Glu
+D-set
FIG. 11. TTX selectively reduces the cGMP response to NMDA
in a o-serine-sensitivemanner. The data show cGMP levels in adult slices exposed to AMPA (30 p M ) , kainate (30 f l ) ,NMDA (300 pM) with and without o-serine (100 pM), and glutamate (3 mM, 2min exposure), in the absence or presenceof 1 pM TTX. “p < 0.001; n = 4-8.
=8
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[Glutamatel (mM1 FIG. 12. Effect of MK-801 (10 p M ) on cGMP levels in 8-day-old (a) and adult (b) rat cerebellar slices exposed for 2 min to a range
of concentrationsof glutamate (n = 4-8). The inset (b) shows the dose-inhibition curve for MK-801(logarithmof molar concentrations on the abscissa) against the cGMP response to 300 pM NMDA in adult slices (pmol/mg of protein on the ordinate; n = 4). In the 8day-old slices, 10 pM MK-801 reduced the cGMP response to 100 pM NMDA from 175 f 4 to 0.3 k 0.1 pmol/mg of protein (n = 4). cGMP levels at all glutamate concentrations tested were significantly reduced by MK-801 (p c 0.05-0.001).
activation in the adult, like in the immature tissue (Garthwaite et al., 1988, 1989u,b),are secondary to the formation of the potent guanylate cyclase activator, NO, from arginine. Although NO production from arginine has yet to be measured directly in intact brain tissue, a NO synthase enzyme is now known to be present in homogenates of cerebellum and forebrain (Knowles et al., 1989; Bredt and Snyder, 1990) and, in all likelihood, in other brain areas as well (Forstermann et al., 1990). The brain enzyme shows similarities to the one in endothelial cells and the adrenal gland in being competitively inhibited by L-NMMA and NNARG and highly Ca*+-dependent, a property conferred on the enzyme by calmodulin. With adult cerebellum, it is not possible to prepare suspensions of cells with the requisite yield and viability to detect NO by bioassay in the way that was done with the immature
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tissue (Garthwaite et al., 1988).Nevertheless, the Ca2+ dependence of the cGMP responses and the argininesensitive inhibition of them by L-NMMA and NNARG make it highly probable that the arginine-toNO pathway is instrumental. A recent short report further shows that L-NMMA is able to block cGMP elevations induced by EAA agonists in adult mouse cerebellum in vivo (Wood et al., 1990). One difference between the adult and the immature tissue with respect to the pharmacology of the NO system is worthy of note. This was with the inhibitor NNARG. In the immature cerebellum, N-NARG inhibited part (about 50%) of the response to NMDA with very high (low nanomolar) potency (see also East and Garthwaite, 1990), but this component disappeared during development, leaving only the other component (high nanomolar) visible in the adult. This suggests that there may be two NO synthases, one of which has a particularly high affinity for N-NARG and is prominent only during development. The NO synthase in adult rat forebrain has a Ki for N-NARG of 0.4 p M (Knowles et al., 1990), which accords with the ICsoof 0.6 p M determined in adult cerebellar slices in our experiments. Developmental studies of the enzyme have not yet been reported.
EAA receptors and NO/cGMP formation in the immature cerebellum In the immature tissue, NO formation appears to be predominantly under the control of NMDA receptors. Agonists at these receptors are highly efficacious, whereas AMPA and quisqualate give only small cGMP elevations. Moreover, extending previous results using cerebellar cell suspensions (Garthwaite, 1985), the effects of the presumed endogenous agonist, glutamate, even in high applied concentration, are mediated solely through NMDA receptors because they are abolished by MK-80 1. The non-NMDA agonist, kainate, is also capable of eliciting cGMP accumulation through NO formation (Garthwaite et al., 19896), but it is known from electrophysiological studies that kainate-induced currents differ from those of glutamate, AMPA, and quisqualate in being very large and nondesensitizing (Kiskin et al., 1986;Patneau and Mayer, 1990). Kainate thus appears to activate the receptors in an unphysiological way. Accordingly, the cGMP response to kainate, like kainate-induced membrane currents (Ishida and Neyton, 1985), can be potently inhibited by glutamate (Garthwaite et al., 19896). The NMDA receptor-NO synthesiscoupling is active surprisingly early on in development. Glutamate is able to evoke significant (sixfold) cGMP accumulation in the cerebellum of the newly born animal, when no excitatory synapses have yet been formed (Garthwaite and Balazs, 1978), and by 3 days (taking the day of birth to be day I), 50-fold increases can be registered in response to NMDA (see legend to Fig. 9). This is the age at which the excitatory climbing fibres first
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make functional synapseswith Purkinje cells. The other excitatory pathway between mossy fibres and granule cells, and thence to Purkinje cells through the granule cell axons (parallel fibres), does not come into operation collectively until several days later (Crepel, 1974; Puro and Woodward, 1977). During early development, Purkinje cells express some NMDA receptors, but later on their sensitivity to this agonist is reduced substantially (Dupont et al., 1987; Garthwaite et al., 1987).The synaptic activation of Purkinje cells by parallel fibres (Kano et al., 1988; Garthwaite and Beaumont, 1989) and climbing fibres (Hirano, 1990; Knopfel et al., 1990) appears only to involve non-NMDA receptors. In contrast, synapses between mossy fibres and granule cells do show marked NMDA receptor-mediated components (Garthwaite and Brodbelt, 1989, 1990; DAngelo et al., 1990). In the 14-day-old rat, this component is visible even with low-frequency stimulation and in the presence of Mg2+(Garthwaite and Brodbelt, 1989). NMDA receptors also participate at synapses between parallel fibres and inhibitory interneurones (basket and stellate cells) found in the molecular layer (Garthwaite and Beaumont, 1989).In autoradiographic binding studies, the majority of cerebellar NMDA receptors are found in the granule cell layer at all stages of development (Cambray-Deakin et al., 1990; Nielsen et al., 1990). Consistent with these data, several experimental approaches have indicated that it is primarily via receptors on granule cells that NMDA and glutamate act to trigger cGMP synthesis in the immature tissue (Garthwaite and Garthwaite, 1987). The actual cGMP elevations mostly do not occur in granule cells, however. Instead, it seems that the NO formed in granule cells diffuses out to activate guanylate cyclase in neighbouring astrocytes (Garthwaite and Garthwaite, 1987; De Vente et al., 1990) and possibly other cell types. EAA receptors and NO/cGMP during maturation NMDA receptors. NMDA became less effective with age, which accords with reductions in the population depolarizing effect of NMDA on granule cells previously observed in the cerebellum during development (Garthwaite et al., 1987). Some of the loss of response to NMDA appears to reflect a decline in the ambient glycine concentration. This is suggested by our findings that in the adult slices, NMDA-induced cGMP formation was enhanced by D-serine and was inhibited by CNQX in a D-Serine-SenSitiVe manner, whereas in the immature slices, D-serine (present results) and CNQX (Garthwaite et al., 19896) were without significant effect. The apparently suboptimal interstitial glycine levels in the adult slices are unlikely to be an in vitro artifact, because previous studies in adult rat and mouse cerebella have shown that glycine and D-Serine increase NMDA receptor-mediated cGMP formation in vivo (Danysz et al., 1989; Wood et al., 1989). We
EXCITATORY AMINO ACIDS AND NITRIC OXIDE further observed that TTX inhibited the response to NMDA only in the absence of added D-serine. Again, this was seen in the adult (present results), but not in the immature tissue (Garthwaite and Garthwaite, 1987). This implies that part of the efficacy of NMDA in the adult is due to the release of an endogenous ligand for the glycine site (presumably glycine itself) by a process that depends on action potential generation. Plausibly, the mechanism entails activation of Golgi cells and a subsequent release of glycine from their axon terminals, which contain rich stores of this amino acid (Ottersen et al., 1988). Appropriately, these terminals are found in the vicinity of dendrites of granule cells, which are major sites of action of NMDA (see above). The loss of sensitivity to NMDA is explained only partly, however, by changes in glycine levels, and other factors must also be involved. One such factor could be increased desensitization taking place during the long applications used. During the synaptic activation of granule cells induced by stimulation of mossy fibres, a large component mediated by NMDA receptors can be observed readily in adult cerebellum (Garthwaite and Brodbelt, 1989, 1990). Furthermore, in adult mouse cerebellum in vivo, both “basal” cGMP levels and the increases that occur in response to pharmacological enhancement of excitatory pathways can be inhibited by competitive or noncompetitive NMDA antagonists (Wood et al., 1982, 1987). Consistent with these results, we find that the basal cGMP levels in adult slices are reduced substantially in the presence of AP5, L-NMMA, or TTX, suggesting that there is endogenous transmitter being released as a result of activity in the neuronal pathways and that the transmitter stimulates NO synthesis by acting on NMDA receptors. Thus, the developmental decline in responsiveness to exogenous NMDA observed in the present experimentsdoes not reflect a genuine loss of functional importance of NMDA receptors at the level of the synapse, either in terms of excitation or as mediators of NO synthesis. AMPA/quisqualate receptors. AMPA and quisqualate became more effective agonists with maturation. Being CNQX-sensitive, the operative non-NMDA receptors appear to be the same typefs)that directly mediates excitation (AMPA receptors). Hence, metabotropic receptor activation by quisqualate and subsequent activation of NO synthase by Ca2+ mobilized from intracellular stores (Watkins et al., 1990) do not appear to occur under the conditions of our experiments. It has been shown recently in binding experiments that the AMPA receptor density increases in both molecular and granule cell layers during development in the rat cerebellum, particularly between the ages of 12 and 20 days (Cambray-Deakin et al., 1990). The concordant profile observed in our experiments may thus reflect an increased receptor density. Interestingly, the regional AMPA receptor density differs
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from that of NMDA receptors, being higher in the molecular layer than in the granule cell layer (CambrayDeakin et al., 1990; Nielsen et al., 1990). Although there is no direct evidence yet about the identity of the cells generating NO in the adult tissue, it is possible that Purkinje or other cells with dendrites in the molecular layer (basket, stellate, and Golgi cells) participate in response to AMPA receptor activation; immunocytochemical studies show that nitroprusside elicits cGMP accumulation in the molecular layer (as well as in the granule cell layer), consistent with there being additional important sites of NO generation there (De Vente et al., 1989, 1990). Furthermore, it is possible that not only neurones, but astrocytes as well, can generate NO (Murphy et al., 1990), and so an involvement of these nonneuronal cells cannot be excluded either. Although it is not yet known if AMPA receptors play a role in the regulation of cGMP levels in vivo, it is interesting that part of the response to glutamate in the adult was insensitive to MK-801 and, therefore, is likely to involve a mechanism other than NMDA receptor activation. It is tempting to ascribe this to stimulation of AMPA receptors, but without an appropriate noncompetitive antagonist, we were not able to test this idea. Concluding comments There remain many unknowns about the NO/cGMP messenger system in the CNS, not the least of which are its functions. In order to provide a framework for the appropriate experiments, it is necessary to define the conditions under which the system becomes activated and to identify the participating cells. The present experiments were directed primarily toward the former objective, and the results reinforce the idea that NMDA receptor activation by the excitatory neurotransmitter (presumably glutamate) is a major trigger for NO formation in both the developing and mature cerebellum. We have found recently that NMDA stimulates cGMP formation in adult and immature hippocampal slices in a manner which is sensitive to NO synthase inhibitors (East and Garthwaite, 1991), suggesting that the mechanism has significance outside the cerebellum. It follows that the NO system is likely to function in an activity-dependent manner, being relatively dormant during low-frequency synaptic transmission, but becoming increasingly activated as the intensity of excitatory transmission increases. This is because of the wellknown voltage dependence of NMDA receptor channels, through which the Ca2+entry needed to activate NO synthase is presumed to occur. The same Ca2+ entry is believed to be the trigger for changes in synaptic efficacy and for the organization of afferent inputs with respect to their target cells during development (Collingridge and Singer, 1990). It is an attractive possibility that a freely diffusible molecule like NO mediates the cell-cell interactions required for these events (Gally et al., 1990).
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Acknowledgment: We thank the Medical Research Council
(U.K.) for financial support, Denice Bradley a n d Geoffrey Williams for valuable assistance, a n d the Wellcome Research Laboratories and Merck, Sharpe a n d D o h m e for providing L-NMMA a n d MK-80 1, respectively.
REFERENCES Biggio G. and Guidotti A. (1976) Climbing fiber activation and 3’3‘cyclic guanosine monophosphate (cGMP) content in cortex and deep nuclei of cerebellum. Bruin Res. 107, 365-373. Bredt D. S.and Snyder S. H.(1990) Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc. Nail. Acad. Sci. USA 87,682-685. Cambray-Deakin M. A., Foster A. C., and Burgoyne R. D. (1990) The expression of excitatory amino acid binding sites during neuritogenesis in the developing rat cerebellum. Dev. Bruin Res. 54,265-27 I. Collingridge G. L. and Singer W. (1990) Excitatory amino acid receptors and synaptic plasticity. Trends Pharmacol. Sci. 11,290296. Crepel F. (1974) Excitatory and inhibitory processes acting upon cerebellar Purkinje cells during maturation in the rat: influence of hypothyroidism. Exp. Brain Res. 20, 403-420. D’Angelo E., Rossi P., and Garthwaite J. (1990) Dual-component NMDA receptor currents at a single central synapse. Nufure 346,467-470. Danysz W., Wroblewski J. T., Brooker G., and Costa E. ( I 989) Modulation of glutamate receptors by phencyclidine and glycine in the rat cerebellum: cCMP increase in vivo. Brain Res. 479, 270276. De Vente J., Bol J . G. J. M., and Steinbusch H. W. M. (1989) Localization of cGMP in the cerebellum of the adult rat: an immunohistochemical study. Brain Res. 504, 332-337. De Vente J., Bol J. G. J. M., Berkelmans H. S., Schipper J., and Steinbusch H. M. W. (1990) Immunocytochemistry of cGMP in the cerebellum of the immature, adult, and aged rat: the involvement of nitric oxide. A micropharmacological study. Eur. J . Neurosci. 2, 845-862. Dupont J.-L., Gardette R., and Crepel F.(1987) Postnatal develop ment of the chemosensitivity of rat cerebellar Purkinje cells to excitatory amino acids: an in vitro study. Dev. Brain Res. 34, 59-68. East S. J . and Garthwaite J . (1990) Nanomolar fl-nitroarginine inhibits NMDA-induced cyclic GMP formation in rat cerebellum. Eur. J. Phurmucol. 184, 3 I 1-3 13. East S.J. and Garthwaite J. (1991) NMDA receptor activation in rat hippocampus induces cyclic GMP formation through the L-arginine-nitric oxide pathway. Neurosci. Lett. 123, 17- 19. Forstermann U., Gorsky L. D., Pollock J. S., Schmidt H. H. H. W., Heller M., and Murad F. (1990) Regional distribution of EDRF/ NO-synthesizing enzymds) in rat brain. Biochem. Biophys. Res. Cornmun. 168,727-732. Gally J. A., Montague P. R., Reeke G. N. Jr., and Edelman G. M. (1990) The NO hypothesis: possible effects of a short-lived, r a p idly diffusible signal in the development and function of the nervous system. Proc. Nail. Acad. Sci. USA 87, 3547-3551. Garthwaite G., Yamini B. Jr., and Garthwaite J. (1987) Selective loss of Purkinje and granule cell responsiveness to N-methyl-Daspartate in rat cerebellum during development. Do.Bruin Res. 36,288-292. Garthwaite J. (1982) Excitatory amino acid receptors and guanosine 3’,5‘-cyclicmonophosphate in incubated slices of immature and adult rat cerebellum. Neuroscience 7, 249 1-2497. Garthwaite J. (1985) Cellular uptake disguises action of L-glutamate on N-methyl-Gaspartate receptors. Br. J. Pharmacol. 85,297307. Garthwaite J. and Balazs R. (1978) Supersensitivity to the cyclic GMP response to glutamate during cerebellar maturation. Nature 275, 328-329.
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Garthwaite J. and Beaumont P. S. (1989) Excitatory amino acid receptors in the parallel fibre pathway in rat cerebellar slices. Neurosci. Lett. 107, 151-156. Garthwaite J. and Brodbelt A. R. (1989) Synaptic activation of Nmethyla-aspartate and non-N-methyl-D-aspartate receptors in the mossy fibre pathway in adult and immature rat cerebellar slices. Neuroscience 29, 40 1-4 12. Garthwaite J. and Brodbelt A. R. (1990) Glutamate as the principal mossy fibre transmitter in rat cerebellum: pharmacological evidence. Eur. J. Neurosci. 2, 177-180. Garthwaite J . and Garthwaite G. (1987) Cellular origins of cyclic GMP responses to excitatory amino acid receptor agonists in rat cerebellum in vitro. J. Neurochem. 48, 29-39. Garthwaite J., Charles S. L., and Chess-Williams R. (1988) Endothelium-derived relaxing factor release on activation of NMDA receptors suggests role as intercellular messenger in the brain. Nature 336, 385-388. Garthwaite J . , Garthwaite G., Palmer R. M. J., and Moncada S. ( 1 989a) NMDA receptor activation induces nitric oxide synthesis from arginine in rat brain slices. Eur. J. Pharmacol. 172, 4 13416. Garthwaite J., Southam E., and Anderton M. (1989b) A kainate receptor linked to nitric oxide synthesis from arginine. J. Neurochem. 53, 1952-1954. Hirano T. (I 990) Synaptic transmission between rat inferior olivary neurons and cerebellar Purkinje cells in culture. J. Neurophysiol. 63, 181-189. Ishida A. T. and Neyton J. (1985) Quisqualate and L-glutamate inhibit retinal horizontal-cell responses to kainate. Proc. Natl. Acad. Sci. USA 82, 1837-1841. Johnson J. W.and Ascher P. (1987) Glycine potentiates the NMDA response in cultured mouse brain neurones. Nature 325, 529531. Kano M., Kato M., and Chang H. S. (1988) The glutamate receptor subtype mediating parallel fibre-Purkinje cell transmission in rabbit cerebellar cortex. Neurosci. Res. 5, 325-337. Kiskin N. I., Krishtal 0. A., and Tsyndrenko A. Y.(1986) Excitatory amino acid receptors in hippocampal neurons: kainate fails to desensitize them. Neurosci. Lett. 63,225-230. Kleckner N.W. and Dingledine R. (1988) Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes. Science 241, 835-837. Knopfel T., Audinat E., and Gahwiler B. H. (1990) Climbing fibre responses in olivo-cerebellar slice cultures. 1. Microelectrode recordings from Purkinje cells. Eur. J. Neurosci. 2, 726-732. Knowles R. G., Palacios M., Palmer R. M. J., and Moncada S. (1989) Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for stimulation of the soluble guanylate cyclase. Proc. Nutl. Acad. Sci. USA 86, 5 159-5 162. Knowles R. G., Palacios M., Palmer R. M. J., and Moncada S.(1990) Kinetic characteristics of nitric oxide synthase from rat brain. Biochem. J. 269, 207-210. Lundberg D. B. A., Breese G. R., Mailman R. B., Frye G. D., and Mueller R. A. (1979) Depression of some drug-induced in vivo changes of cerebellar guanosine 3‘,5’-monophosphateby control of motor and respiratory responses. Mol. Pharmacol. 15, 246256. Mayer M. L.,Vyklicky L., and Clements J. (1989) Regulation of NMDA receptor desensitization in mouse hippocampal neurons by glycine. Nature 338, 425-427. Murphy S..Minor R. L. Jr., Welk G., and Hamson D. G. (1990) Evidence for an astrocyte-derived vasorelaxing factor with properties similar to nitric oxide. J. Neurochem. 55, 349-351. Nielsen E. O.,Drejer J., Cha J.-H. J., Young A. B., and Honor6 T. ( 1990) Autoradiographic characterization and localization of quisqualate binding sites in rat brain using the antagonist [’H]6cyano-7-nitroquinoxaline-2,3-dione:comparison with (R,S)[3H]a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid binding sites. J. Neurochem. 54, 686-695. Ottersen 0.P., Storm-Mathisen J., and Somogyi P. (1988) Colocalization of glycine-like and GABA-like immunoreactivities in
EXCITATORY AMINO ACIDS AND NITRIC OXIDE Golgi cell terminals in the rat cerebellum: a postembedding light and electron microscopic study. Brain Res. 450, 342-353. Patneau D. K. and Mayer M. L. (1990) Structure-activity relationships for amino acid transmitter candidates acting at N-methylD-aspartate and quisqualate receptors. J. Neurosci 10, 23852399. Puro D. G. and Woodward D. J. ( 1977) Maturation of evoked mossy fiber input to rat cerebellar Purkinje cells (11). Exp. Bruin. Res. 28,427-44 I . Rubin E. H. and Ferrendelli J. A. (1977) Distribution and regulation of cyclic nucleotide levels in cerebellum, in vivo. J. Neurochem. 29,43-5 I . Watkins J. C., Krogsgaard-LarsenP., and Honor6 T. (1990) Structureactivity relationships in the development of excitatory amino acid receptor agonists and competitive antagonists. Trends fharmacol. Sci 1 I, 25-33. Wood P. L., Richard J. W., Pilapil C., and Nair N. P. V. (1982) Antagonists of excitatory amino acids and cyclic guanosine
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monophosphate in cerebellum. Neuropharmacology 21, 12351238. Wood P. L., Steel D., McPherson S. E., Cheney D. L., and Lehmann J. (1 987) Antagonism of N-methyl-o-aspartate (NMDA) evoked increases in cerebellar cGMP and striatal ACh release by phencyclidine (PCP) receptor agonists: evidence for possible allosteric coupling of NMDA and PCP receptors. Can. J. fhysiol. fharmacol. 65, 1923-1927. Wood P. L., Emmett M. R., Rao T. S., Mick S., Cler J., and Iyengar S. (1989) In vivo modulation of the N-methyl+-aspartate receptor complex by o-serine: potentiation of ongoing neuronal activity as evidenced by increased cerebellar cyclic GMP. J. Neurochem. 53,979-98 1. Wood P. L., Emmett M. R., Rao T. S., Cler J., Mick S., and Iyengar S. (1990) Inhibition of nitric oxide synthase blocks N-methylD-aspartate-, quisqualate-, kainate-, hannaline-, and pentylenetetrazol-dependent increases in cerebellar cyclic GMP in vivo. J. Neurochem. 55,346-348.
J. Neurochem., Vol. 56. No. 6, 1991
Raven Press, Ltd., New York 0 1991 International Society for Neurochemistry
Excitatory Amino Acid Receptors Coupled to the Nitric Oxide/Cyclic GMP Pathway in Rat Cerebellum During Development E. Southam, S. J. East, and J. Garthwaite Department of Physiology,University of Liverpool, Liverpool, England
preparations by measuring cyclic GMP accumulation. In the immature tissue, N-methyl-D-aspartate(NMDA) and glutamate were highly efficacious agonists, whereas a-amino-3hydroxy-5-methyl-4-isoxazolepropionate(AMPA) and quisqualate evoked only small responses. The effect of glutamate at all concentrations tested (up to 10 mM) was abolished by the NMDA antagonist, (+)-5-methyl-l0,1I-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-iminemaleate (MK-80I). In adult slices, AMPA and quisqualate were much more effective and their effects were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist for ionotropic non-NMDA receptors, whereas the apparent efficacy of NMDA was greatly reduced. The major changes took place between 8 and 14 days postnatum and, in the case of NMDA, part of the loss of sensitivity appeared to reflect a decline in the ambient levels of glycine with age. Moreover, a component of the response to glutamate in the adult was resistant to MK-80 I .
Cyclic GMP accumulations induced by NMDA and nonNMDA agonists alike were Ca’+-dependent and could be antagonized by competitive NO synthase inhibitors in an arginine-sensitive manner, indicating that they are all mediated by NO formation. With one of the inhibitors, L - N ~ nitroarginine, a highly potent component (ICso= 6 M)evident in slices from rats of up to 8 days old was lost during maturation, indicating that there may be a NO synthase isoform which is prominent only in the immature tissue. Cyclic GMP levels in adult slices under “basal” conditions were reduced markedly by blocking NMDA receptors, by inhibiting action potentials with tetrodotoxin, or by NO synthase inhibition, suggesting that the endogenous transmitter released during spontaneous synaptic activity acts mainly through NMDA receptors to trigger NO formation. Key Words: Glutamate-N-Methyl-D-aspartate receptors-Nitric oxide-Cyclic GMP-Cerebellum-Development. Southam E. et al. Excitatory amino acid receptors coupled to the nitric oxide/cyclic GMP pathway in rat cerebellum during development. J. Neurochem. 56,2072-208 I (1 99 1).
Studies on slices and dispersed cells from the cerebellum of immature rats have shown that activation of excitatory amino acid (EAA) receptors triggers the Ca2+-dependent synthesis of the novel intercellular messenger, nitric oxide (NO), from L-arginine (Garthwaite et al., 1988, 1 9 8 9 ~ )A . major action of the NO so formed is to stimulate the enzyme guanylate cyclase and thus trigger cyclic G M P (cGMP) synthesis. In the immature tissue, this effect of glutamate appears to be mediated mostly, if not entirely, through the N-methylD-aspartate (NMDA) class of receptor (Garthwaite, I985), even though the non-NMDA receptor agonist, kainate, is also able to evoke, through NO, a large ac-
cumulation of cGMP (Garthwaite et al., 1989b). The other selective non-NMDA receptor agonists, quisqualate and c~-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), even at high concentration, induce only small cGMP elevations. The question inevitably arises as to the relevance of these findings to the adult. During the postnatal development of the cerebellum, changes take place in the relative depolarizing potency and efficacy of selective glutamate receptor agonists. In particular, there is an apparent loss of chemosensitivity to NMDA of both principal neuronal types, i.e., granule cells and Purkinje cells (Dupont et al., 1987; Garthwaite et al., 1987).
Received September 24, 1990; revised manuscript received November 25,1990; accepted December 5, 1990. Address correspondence and reprint requests to Dr. J . Garthwaite at Department of Physiology, University of Liverpool, Brownlow Hill, P.O. Box 147, Liverpool L69 3BX, U.K. A bbreviarinns used: AMPA, ol-amino-3-hydroxy-5-methyl-4-isox-
azolepropionate; AP5, D-2-amino-5-phosphonopentanoate; cGMP, EAA, cyclic GMP; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; excitatory amino acid; MK-80 1, (+)-5-methyl-l0,1l-dihydro-5H-dibenzo[a,d]cyclohepten-5,I0-iminemaleate; N-NARC, L-NG-nitroarginine; NMDA, N-methyl-D-aspartate; L-NMMA, L-p-monomethyl-L-arginine; NO, nitric oxide; TTX, tetrodotoxin.
Abstract: The coupling of excitatory amino acid receptors to the formation of nitric oxide (NO) from arginine during the postnatal development of rat cerebellum was assayed in slice
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homogenized by sonication. The protein content of the homogenate was determined by the automated Lowry method and, following centrifugation, the cGMP content of the supernatant was measured by radioimmunoassay (see Garthwaite and Garthwaite, 1987). Results are expressed as mean cGMP levels per milligram of protein (or percentage of control levels) -t SEM, and statistical comparisons were made using the Student's t test; n values refer to the number of slices used and, within any experimental group, each slice was from a different animal. In experiments requiring a Caz+-freemedium, slices were transferred, 15 min before the addition of test compounds, to a solution in which CaC12 had been replaced by 1 mM EGTA. The same preincubation period was adopted when antagonists acting at the receptors or on the NO synthase were tested.
Nevertheless, in the adult cerebellum in vivo, activation of afferent excitatory pathways is known to give rise to large elevations in cerebellar cGMP levels (Biggio and Guidotti, 1976; Rubin and Ferrendelli, 1977; Lundberg et al., 1979; Wood et al., 1982). Moreover, the NOsynthesizing enzyme, NO synthase, is known to be present in adult rat brain (Knowles et al., 1989; Bredt and Snyder, 1990; Forstermann et al., 1990). Accordingly, the aim of the present study was to investigate the coupling of EAA receptors to the NO/ cGMP system in adult rat cerebellum and, in the light of differences observed in comparison with the immature tissue, to chart the changes that occur during the maturation of this brain region.
Materials
MATERIALS AND METHODS
AMPA, D-2-amino-5-phosphonopentanoic acid (APS), and 6-cyano-7-nitroquinoxaline-2,3-dione(CNQX) were from Tocris Neuramin; kainic acid, NMDA, quisqualic acid, LNG-nitroarginine(N-NARG), sodium nitroprusside, and tetrodotoxin (TTX) were from Sigma; (+)-5-methyl-10,l l-dihydro-5H-dibenzo[a,d]cyclohepten-5,lO-iminemaleate (MK-801) was from Merck, Sharpe and Dohme; and L-NGmonomethyl-L-arginine (L-NMMA) was supplied by the Wellcome Research Laboratories.
Methods Rats (Wistar origin, either sex) were killed by decapitation. For animals aged 21 days and over, the cerebellum was excised and the hemispheres removed by parasagittal razor cuts. One of the cut surfaces of the vermis was glued (with cyanoacrylate) onto the stage of a Vibroslice (Campden Instruments) and then embedded in 3% agar (made up in 0.9% saline and kept stirred at 37°C). The agar formed an exoskeleton which greatly enhanced the stability of the tissue during sectioning. Slices were cut at 400-pm intervals in cool ( 1 2- 14°C) Krebs-Henseleit solution containing (in mM) NaCl ( 120), KCI (2),CaC12(2),MgS04( I . 19),KH2P04(1.1 8), NaHC03 (26), and glucose ( I l), gassed with 5% C02in 02. pH 7.4. Subsequently, the slices were allowed to recover for about 30 min in gassed solution maintained at 37°C in a shakingwater bath. The slices were then redistributed so that each incubation vessel contained slices (usually four) from different animals, and the preincubation was continued for about an additional hour. For younger animals, slices of vermis were cut in the parasagittal plane using a tissue chopper (see Garthwaite and Garthwaite, 1987), but the procedures otherwise were the same. At predetermined times after the addition of test compounds, slices were quickly removed from the incubating medium and inactivated for 3 min in 200 pI of boiling 50 mMTris, 4 mMEDTA buffer, pH 7.6. The slices were then 90
la)
0
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.-
80
cGMP responses to EAA receptor agonists The mean cGMP level in unstimulated adult (25week-old) cerebellar slices was 1.75 k 0.13 pmol/mg of protein (n = 85). Addition of the non-NMDA agonists, kainate, AMPA, and quisqualate, evoked a prompt rise in c G M P levels which peaked within 30 s, whereas the response to NMDA was slower (Fig. la). Subsequent experiments used a 2-min exposure for NMDA and 30-s exposures for the others. Concentration-response curves (Fig. 1b) showed that the apparent efficacy of kainate was higher than that of AMPA, the responses at or near their maximally effective concentrations (100 p M ) being about 80 and
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FIG. 1. Time courses (a) and concentration-response curves (b)for cGMP levels in adult rat cerebellar slices exposed to kainate (O), AMPA (0),quisqualate (O), and NMOA (m), each at a concentration of 100 pA-4 (n = 3-4).
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50 pmol/mg of protein, respectively. The corresponding ECSo values were approximately 20 and 10 pM. The effects of NMDA and quisqualate were also concentration-dependent, but no clear maxima were obtained: at the highest concentrations tested ( 100 p M quisqualate, 1 M N M D A ) , cGMP levels were similar to those observed with 100 pM AMPA. Developmental changes. In terms of absolute cGMP levels, the response to kainate, at a concentration (100 p M ) which is maximally effective in adult and immature tissues (Garthwaite, 1982),remained relatively constant throughout development (see legend to Fig. 2), and so this was used as the reference. In agreement with previous findings(Garthwaite, 1982),NMDA (100 p M ) evoked a very large accumulation of cGMP in slices from 8-day-old animals (Fig. 2), the levels attained being about twice those produced by kainate. However, a striking reduction occurred during maturation, particularly between 8 and 14 days, so that in the adult, the response to 100 pM NMDA was only 10%of that to kainate. In contrast, the effect of AMPA (100 p M ) became larger during development (20% of the response to kainate at 8 days rising to 70% in the adult), the major change again taking place between 8 and 14 days. Quisqualate (100 p M ) , though starting from a lower base, behaved similarly to AMPA (Fig. 2). One factor that could contribute to the loss of response to NMDA is a decline in the ambient levels of glycine. Glycine binds with high affinity to a site on the NMDA receptor, and occupation of this site is necessary for opening of the NMDA receptor channel (Johnson and Ascher, 1987; Kleckner and Dingledine, 1988);glycine also may inhibit a component of NMDA receptor desensitization (Mayer et al., 1989). DSerine 250
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Age (days) FIG. 2. Developmental changes in cGMP responses to kainate
(0, AMPA (0), quisqualate (O),and NMDA (m), all at 100 pM. The data are expressed as percent mean response to 100 pM kainate at each age; the absolute values for these reference responses were 87 k 6, 114 & 4, 71 k 5, and 70 f 3 pmol/mg of protein at 8, 14, 21, and >30 days, respectively (n = 4-8).
J. Neurochem., Val. S6. No. 6, 1991
is an agonist for this glycine site but, unlike glycine itself, is not transported into cells nor does it activate inhibitory glycine receptors. The dose-response curve for NMDA was not affected significantly by D-serine in slices from 8-day-old animals but, in the adult, cGMP accumulations at 100 and 300 pMNMDA were significantlylarger (Fig. 3). Developmentally, this augmentation by D-serine became evident at 21 days of age (Fig. 4). Effect oJCNQX. CNQX is generally regarded as a fairly selective non-NMDA antagonist, but because of its activity on the glycine site of the NMDA receptor, it can also inhibit NMDA responses when glycine levels are low (Watkins et al., 1990).In the immature (8-dayold) cerebellum, CNQX inhibits the cGMP response to kainate, but not that to NMDA (Garthwaite et al., 19896). In slices of the adult, however, CNQX inhibited not only responses to kainate, AMPA, and quisqualate (each at 30 p M ) , but also that to NMDA (300 pM), the ICsOvalues (2-4 p M ) being similar for all agonists (Fig. 5). The inhibition by CNQX of the NMDA response, but not of the response to AMPA, could be nullified, however, by including D-Serine in the incubation medium (Fig. 6). Involvement of NO Addition of L-arginine, the substrate for the NO synthase enzyme, increased the cGMP response to AMPA in adult cerebellar slices in a dose-dependent manner (Fig. 7). The effects were maximal (50% increase) at 100-300 pM arginine and then declined. The analogue, L-NMMA (100 p M ) , which is a competitive inhibitor of the formation of NO from arginine (Knowles et al., 1990), markedly reduced the responses to AMPA, quisqualate, kainate, and NMDA (Fig. 8). The ICsOvalue for L-NMMA against AMPA was about 6 pM(data not shown), and its inhibitory effects against all agonists could be counteracted by additional arginine (Fig. 8). Another NO synthase inhibitor, N-NARG, is a more potent inhibitor than L-NMMA of the NMDA-induced cGMP formation in slices of immature cerebellum, where it exhibits a strongly biphasic inhibition curve (ICso = 6 and 600 nM,Fig. 9a; East and Garthwaite, 1990). In the adult tissue, by contrast, the curve was a simple monophasic one (ICs0= 600 nM; Fig. 9a). During development, the loss of the higher potency component was virtually complete by 15 days (Fig. 9b). The cGMP elevations produced by NMDA and nonNMDA receptor agonists alike were Ca2+-dependent: preincubating adult slices in Ca2+-freemedium for 15 min reduced the subsequent response to 300 pM NMDA from 28 k 3 to 2.1 4 0.6, the response to 100 pM AMPA from 45 2 2 to 1.4 f 0.3, and the response to 30 pMquisqualate from 24 f 2 to 2.2 +- 0.5 pmol/ mg of protein (all p < 0.00 1, n = 4). A compound which releases NO spontaneously, sodium nitroprusside (100 pM, 5-min exposure) raised cGMP levels in adult slices to 65 f 3 pmol/mg of pro-
p
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EXCITATORY AMINO ACIDS AND NITRIC OXIDE 200
*.m controls 0.0
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lCNQXl [pM) FIG. 5. Inhibitionby CNQX of the cGMP elevations induced by 30 FIG. 3. Concentration-cGMP response relationships for NMDA in cerebellar slices from 8-day-old (circles) and adult (squares) rats in the absence (filled symbols) and presence (open symbols) of 100 f l D-serine. Responses were significantly increased by the amino acid only at 100 jtM and 300 NMDA in the adult ( p < 0.05 and 0.001, respectively; n = 4-12).
tein (n = 8). In immature (8-day-old) slices, the corresponding increase was to 105 t 12 pmol/mg of protein (n = 4). Maintenance of basal cGMP levels and effect of n x In slices of the adult, unstimulated levels of cGMP were reduced by either L-NMMA (100 F M ) or TTX (1 p M ) to 20-30% of controls. Also, CNQX and the NMDA antagonist, AP5, each lowered the levels by 50%, but the inhibition by CNQX could be overcome by adding D-serine (Fig. 10).
jtM concentrationsof kainate(O), AMPA (0),and quisqualate (O), and by 300 pVl NMDA (m) in adult cerebellar slices (n = 3-4).
We also tested whether the increases in cGMP induced by receptor agonists were sensitive to TTX in the adult tissue (Fig. 11). This proved not to be the case for AMPA and kainate (and also glutamate); however, with NMDA, cGMP levels were reduced by about 40%. No such inhibition by TTX was observed in the presence of D-serine.
8o 6o
1 1
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*+ T
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agonrst
+CNBX
tCNBX +D-serine tD-serine
5
9
14
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>30
Age (days1
FIG. 4. Developmentalchanges in the enhancing effect of D-serine (100 pM) on cGMP responses to 300 jtM NMDA (*p < 0.002; '*p < 0.001; n = 4-12).
FIG. 6. In adult slices, o-serine (100 p.M) does not affect the inhibition by CNQX (10 f l )of the cGMP response to 30 jtM AMPA (a), but counteracts that to 300 f l NMDA (b). In the absence of D-serine, CNQX significantly inhibited responses to both NMDA (p < 0.01)and AMPA (p < 0.001). o-Serinealone selectively increased the response to NMDA ( p < 0.001). With o-serine present, the degree of inhibition by CNQX of the response to AMPA was similar ( p < 0.001), whereas that to NMDA was nullified (n = 4).
J. Neurochem.. Vol. 56. No. 6, 1991
20 76
E. SOUTHAM ET AL. 90 -I 125
8 2
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FIG. 7. L-Arginine augments cGMP response to AMPA (100 &f)
in adult cerebellar slices in a dosedependent manner. Significant changes occurred at arginine concentrationsof 30 pM and above ( p < 0.05-0.001; n = 6-8).
Actions of glutamate Glutamate increased cGMP levels in slices of both the 8-day-old and adult in a dose-dependent manner (Fig. 12). Because of the presence of uptake mechanisms, it is necessary to use high concentrations of glutamate for it to penetrate the tissue extracellular spaces; this, in turn, makes it very difficult to study the pharmacology of its effects with competitive antagonists like CNQX and AP5, because correspondingly high antagonist concentrations are needed and then their selectivity becomes questionable (Garthwaite, 1985). Ac-
AMPA
Quis
Kainate
NMDA
0 agonist
@
+100pM L-NMMA +L-NMMA +100pM L-arg +L-NMMA +300pM L-arg
FIG. 8. L-NMMA (100 pA4) inhibits cGMP responses to 100 pM
AMPA, quisqualate, kainate, and NMDA, and this inhibitioncan be neutralized by additionalarginine, becominginsignificantwith 300 phd arginine (see key; n = 4).
J. Neuroehem., Vol. 56, No. 6, 1991
1
-
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11
Age ldsys)
15
31
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FIG. 9. a: Inhibition of cGMP responses to 300 pM NMDA by N-
NARG in adult (m)and 8-day-old (0)slices showing, only in the latter, a component sensitive to low nanomolar concentrationsof N-NARG. Values for the control responses were 35 ? 4 (adult, n = 7) and 161 f 19 (8 days, n = 11) pmol of cGMP/mg of protein. For the other data, n = 3-1 7. Displayed in b are the developmental changes in the inhibitory effects of 100 nM and 3 pM N-NARG, showing loss of the high potency component by 15 days of age (note logarithmic scale). The control responses to 300 pM NMDA were as follows (in pmol of cGMP/mg of protein): 46 f 6 (3 days), 115 k 7 (5days), 161 5 19 (8 days), 89 k 9 (11 days), 78 k 6 (15 days), and 35 k 4 (29-33 days) (n = 4-12).
cordingly, we used the noncompetitive NMDA antagonist, MK-801, to determine the extent to which NMDA receptors contribute to glutamate-induced cGMP formation. In 8-day-old cerebellar slices, the effects of glutamate at all concentrations tested were abolished by MK-80 1 at a dose (10 p M ) which completely inhibited the cGMP response to NMDA (Fig. 12a). In the adult slices, however, a component remained in the presence of MK-801 (Fig. 12b). This MK-80 1-resistant portion of the response became larger as the glutamate concentration was raised (36% at 1 mM, increasing to 68% at 10 mM). DISCUSSION Because of the experimental advantages offered, most of the previous work dealing with the coupling of EAA receptors to NO synthesis has been camed out
EXCITATORY AMINO ACIDS AND NITRIC OXIDE
p
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FIG. 10. NMDA receptor activation and NO formation sustain
“basal” cGMP levels in adult cerebellar slices. The first column (a) corresponds to levels in control slices (set at 100%; absolute value = 2.94 f 0.16 pmol of cGMP/mg of protein; n = 14). In the other columns, the slices were incubated with the following: b, CNQX (10 pM, n = 4); c, AP5 (50pM, n = 8); d, CNQX plus AP5 (n = 4); e, L-NMMA (100 f l ,n = 8); f, TTX (1 pM, n = 8); g, o-serine (100 pM, n = 4); h, CNQX plus o-serine (n = 4). *p < 0.05,**p < 0.01 compared to controls.
-.-c
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on preparations of the immature cerebellum, and the question inevitably arose as to the relevance of the findings to the adult. The present study finds some basic similarities, but also some differences that could have a significant bearing on our present understanding of the physiological regulation of this novel messenger system. NO as the mediator of cGMP responses The first conclusion to be made is that the cGMP elevations taking place in response to EAA receptor
AMPA Kainate NMDA NMDA
Glu
+D-set
FIG. 11. TTX selectively reduces the cGMP response to NMDA
in a o-serine-sensitivemanner. The data show cGMP levels in adult slices exposed to AMPA (30 p M ) , kainate (30 f l ) ,NMDA (300 pM) with and without o-serine (100 pM), and glutamate (3 mM, 2min exposure), in the absence or presenceof 1 pM TTX. “p < 0.001; n = 4-8.
=8
10
0
0
1
10
[Glutamatel (mM1 FIG. 12. Effect of MK-801 (10 p M ) on cGMP levels in 8-day-old (a) and adult (b) rat cerebellar slices exposed for 2 min to a range
of concentrationsof glutamate (n = 4-8). The inset (b) shows the dose-inhibition curve for MK-801(logarithmof molar concentrations on the abscissa) against the cGMP response to 300 pM NMDA in adult slices (pmol/mg of protein on the ordinate; n = 4). In the 8day-old slices, 10 pM MK-801 reduced the cGMP response to 100 pM NMDA from 175 f 4 to 0.3 k 0.1 pmol/mg of protein (n = 4). cGMP levels at all glutamate concentrations tested were significantly reduced by MK-801 (p c 0.05-0.001).
activation in the adult, like in the immature tissue (Garthwaite et al., 1988, 1989u,b),are secondary to the formation of the potent guanylate cyclase activator, NO, from arginine. Although NO production from arginine has yet to be measured directly in intact brain tissue, a NO synthase enzyme is now known to be present in homogenates of cerebellum and forebrain (Knowles et al., 1989; Bredt and Snyder, 1990) and, in all likelihood, in other brain areas as well (Forstermann et al., 1990). The brain enzyme shows similarities to the one in endothelial cells and the adrenal gland in being competitively inhibited by L-NMMA and NNARG and highly Ca*+-dependent, a property conferred on the enzyme by calmodulin. With adult cerebellum, it is not possible to prepare suspensions of cells with the requisite yield and viability to detect NO by bioassay in the way that was done with the immature
J . Neurochem., Vol. 56, No. 6, 1991
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E. SOUTHAM ET AL.
tissue (Garthwaite et al., 1988).Nevertheless, the Ca2+ dependence of the cGMP responses and the argininesensitive inhibition of them by L-NMMA and NNARG make it highly probable that the arginine-toNO pathway is instrumental. A recent short report further shows that L-NMMA is able to block cGMP elevations induced by EAA agonists in adult mouse cerebellum in vivo (Wood et al., 1990). One difference between the adult and the immature tissue with respect to the pharmacology of the NO system is worthy of note. This was with the inhibitor NNARG. In the immature cerebellum, N-NARG inhibited part (about 50%) of the response to NMDA with very high (low nanomolar) potency (see also East and Garthwaite, 1990), but this component disappeared during development, leaving only the other component (high nanomolar) visible in the adult. This suggests that there may be two NO synthases, one of which has a particularly high affinity for N-NARG and is prominent only during development. The NO synthase in adult rat forebrain has a Ki for N-NARG of 0.4 p M (Knowles et al., 1990), which accords with the ICsoof 0.6 p M determined in adult cerebellar slices in our experiments. Developmental studies of the enzyme have not yet been reported.
EAA receptors and NO/cGMP formation in the immature cerebellum In the immature tissue, NO formation appears to be predominantly under the control of NMDA receptors. Agonists at these receptors are highly efficacious, whereas AMPA and quisqualate give only small cGMP elevations. Moreover, extending previous results using cerebellar cell suspensions (Garthwaite, 1985), the effects of the presumed endogenous agonist, glutamate, even in high applied concentration, are mediated solely through NMDA receptors because they are abolished by MK-80 1. The non-NMDA agonist, kainate, is also capable of eliciting cGMP accumulation through NO formation (Garthwaite et al., 19896), but it is known from electrophysiological studies that kainate-induced currents differ from those of glutamate, AMPA, and quisqualate in being very large and nondesensitizing (Kiskin et al., 1986;Patneau and Mayer, 1990). Kainate thus appears to activate the receptors in an unphysiological way. Accordingly, the cGMP response to kainate, like kainate-induced membrane currents (Ishida and Neyton, 1985), can be potently inhibited by glutamate (Garthwaite et al., 19896). The NMDA receptor-NO synthesiscoupling is active surprisingly early on in development. Glutamate is able to evoke significant (sixfold) cGMP accumulation in the cerebellum of the newly born animal, when no excitatory synapses have yet been formed (Garthwaite and Balazs, 1978), and by 3 days (taking the day of birth to be day I), 50-fold increases can be registered in response to NMDA (see legend to Fig. 9). This is the age at which the excitatory climbing fibres first
J. Neurochem., Vol. 56, No. 6. 1991
make functional synapseswith Purkinje cells. The other excitatory pathway between mossy fibres and granule cells, and thence to Purkinje cells through the granule cell axons (parallel fibres), does not come into operation collectively until several days later (Crepel, 1974; Puro and Woodward, 1977). During early development, Purkinje cells express some NMDA receptors, but later on their sensitivity to this agonist is reduced substantially (Dupont et al., 1987; Garthwaite et al., 1987).The synaptic activation of Purkinje cells by parallel fibres (Kano et al., 1988; Garthwaite and Beaumont, 1989) and climbing fibres (Hirano, 1990; Knopfel et al., 1990) appears only to involve non-NMDA receptors. In contrast, synapses between mossy fibres and granule cells do show marked NMDA receptor-mediated components (Garthwaite and Brodbelt, 1989, 1990; DAngelo et al., 1990). In the 14-day-old rat, this component is visible even with low-frequency stimulation and in the presence of Mg2+(Garthwaite and Brodbelt, 1989). NMDA receptors also participate at synapses between parallel fibres and inhibitory interneurones (basket and stellate cells) found in the molecular layer (Garthwaite and Beaumont, 1989).In autoradiographic binding studies, the majority of cerebellar NMDA receptors are found in the granule cell layer at all stages of development (Cambray-Deakin et al., 1990; Nielsen et al., 1990). Consistent with these data, several experimental approaches have indicated that it is primarily via receptors on granule cells that NMDA and glutamate act to trigger cGMP synthesis in the immature tissue (Garthwaite and Garthwaite, 1987). The actual cGMP elevations mostly do not occur in granule cells, however. Instead, it seems that the NO formed in granule cells diffuses out to activate guanylate cyclase in neighbouring astrocytes (Garthwaite and Garthwaite, 1987; De Vente et al., 1990) and possibly other cell types. EAA receptors and NO/cGMP during maturation NMDA receptors. NMDA became less effective with age, which accords with reductions in the population depolarizing effect of NMDA on granule cells previously observed in the cerebellum during development (Garthwaite et al., 1987). Some of the loss of response to NMDA appears to reflect a decline in the ambient glycine concentration. This is suggested by our findings that in the adult slices, NMDA-induced cGMP formation was enhanced by D-serine and was inhibited by CNQX in a D-Serine-SenSitiVe manner, whereas in the immature slices, D-serine (present results) and CNQX (Garthwaite et al., 19896) were without significant effect. The apparently suboptimal interstitial glycine levels in the adult slices are unlikely to be an in vitro artifact, because previous studies in adult rat and mouse cerebella have shown that glycine and D-Serine increase NMDA receptor-mediated cGMP formation in vivo (Danysz et al., 1989; Wood et al., 1989). We
EXCITATORY AMINO ACIDS AND NITRIC OXIDE further observed that TTX inhibited the response to NMDA only in the absence of added D-serine. Again, this was seen in the adult (present results), but not in the immature tissue (Garthwaite and Garthwaite, 1987). This implies that part of the efficacy of NMDA in the adult is due to the release of an endogenous ligand for the glycine site (presumably glycine itself) by a process that depends on action potential generation. Plausibly, the mechanism entails activation of Golgi cells and a subsequent release of glycine from their axon terminals, which contain rich stores of this amino acid (Ottersen et al., 1988). Appropriately, these terminals are found in the vicinity of dendrites of granule cells, which are major sites of action of NMDA (see above). The loss of sensitivity to NMDA is explained only partly, however, by changes in glycine levels, and other factors must also be involved. One such factor could be increased desensitization taking place during the long applications used. During the synaptic activation of granule cells induced by stimulation of mossy fibres, a large component mediated by NMDA receptors can be observed readily in adult cerebellum (Garthwaite and Brodbelt, 1989, 1990). Furthermore, in adult mouse cerebellum in vivo, both “basal” cGMP levels and the increases that occur in response to pharmacological enhancement of excitatory pathways can be inhibited by competitive or noncompetitive NMDA antagonists (Wood et al., 1982, 1987). Consistent with these results, we find that the basal cGMP levels in adult slices are reduced substantially in the presence of AP5, L-NMMA, or TTX, suggesting that there is endogenous transmitter being released as a result of activity in the neuronal pathways and that the transmitter stimulates NO synthesis by acting on NMDA receptors. Thus, the developmental decline in responsiveness to exogenous NMDA observed in the present experimentsdoes not reflect a genuine loss of functional importance of NMDA receptors at the level of the synapse, either in terms of excitation or as mediators of NO synthesis. AMPA/quisqualate receptors. AMPA and quisqualate became more effective agonists with maturation. Being CNQX-sensitive, the operative non-NMDA receptors appear to be the same typefs)that directly mediates excitation (AMPA receptors). Hence, metabotropic receptor activation by quisqualate and subsequent activation of NO synthase by Ca2+ mobilized from intracellular stores (Watkins et al., 1990) do not appear to occur under the conditions of our experiments. It has been shown recently in binding experiments that the AMPA receptor density increases in both molecular and granule cell layers during development in the rat cerebellum, particularly between the ages of 12 and 20 days (Cambray-Deakin et al., 1990). The concordant profile observed in our experiments may thus reflect an increased receptor density. Interestingly, the regional AMPA receptor density differs
2079
from that of NMDA receptors, being higher in the molecular layer than in the granule cell layer (CambrayDeakin et al., 1990; Nielsen et al., 1990). Although there is no direct evidence yet about the identity of the cells generating NO in the adult tissue, it is possible that Purkinje or other cells with dendrites in the molecular layer (basket, stellate, and Golgi cells) participate in response to AMPA receptor activation; immunocytochemical studies show that nitroprusside elicits cGMP accumulation in the molecular layer (as well as in the granule cell layer), consistent with there being additional important sites of NO generation there (De Vente et al., 1989, 1990). Furthermore, it is possible that not only neurones, but astrocytes as well, can generate NO (Murphy et al., 1990), and so an involvement of these nonneuronal cells cannot be excluded either. Although it is not yet known if AMPA receptors play a role in the regulation of cGMP levels in vivo, it is interesting that part of the response to glutamate in the adult was insensitive to MK-801 and, therefore, is likely to involve a mechanism other than NMDA receptor activation. It is tempting to ascribe this to stimulation of AMPA receptors, but without an appropriate noncompetitive antagonist, we were not able to test this idea. Concluding comments There remain many unknowns about the NO/cGMP messenger system in the CNS, not the least of which are its functions. In order to provide a framework for the appropriate experiments, it is necessary to define the conditions under which the system becomes activated and to identify the participating cells. The present experiments were directed primarily toward the former objective, and the results reinforce the idea that NMDA receptor activation by the excitatory neurotransmitter (presumably glutamate) is a major trigger for NO formation in both the developing and mature cerebellum. We have found recently that NMDA stimulates cGMP formation in adult and immature hippocampal slices in a manner which is sensitive to NO synthase inhibitors (East and Garthwaite, 1991), suggesting that the mechanism has significance outside the cerebellum. It follows that the NO system is likely to function in an activity-dependent manner, being relatively dormant during low-frequency synaptic transmission, but becoming increasingly activated as the intensity of excitatory transmission increases. This is because of the wellknown voltage dependence of NMDA receptor channels, through which the Ca2+entry needed to activate NO synthase is presumed to occur. The same Ca2+ entry is believed to be the trigger for changes in synaptic efficacy and for the organization of afferent inputs with respect to their target cells during development (Collingridge and Singer, 1990). It is an attractive possibility that a freely diffusible molecule like NO mediates the cell-cell interactions required for these events (Gally et al., 1990).
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Acknowledgment: We thank the Medical Research Council
(U.K.) for financial support, Denice Bradley a n d Geoffrey Williams for valuable assistance, a n d the Wellcome Research Laboratories and Merck, Sharpe a n d D o h m e for providing L-NMMA a n d MK-80 1, respectively.
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