Journal of Nmrochemisrry Raven Press, Ltd., New York Q 1992 International Society for Neurochemistry

Two Pathways of Cyclic GMP Production Through -Mediated Synthesis

Daisuke Okada

Laboratory for Neural Networks, Frontier Research Program, UIKIiN, Saiiama. Japan

Abstract: The selective for the metabotropic gluta- In a calcium-free medium, ACPD still induced a rise in mate receptor and the ionotropic non-N-methyl-naspar- cGMP level, whereas AMPA did not. When the molecular tate (NMDA) glutamate receptor, (?)-1 -aminocyclopen- layer was isolated to determine the cGMP content sepa- tane-trans- 1,3dicarboxylic acid (ACPD) and (R,S)-a-ami- rately from that in the rest of the cerebellar cortex, it was no- 3 - hydroxy - 5 - meth ylisoxazole-4-propionate (AMPA), found that ACPD raised the cGMP level mainly in the mo- respectively, increased the cyclic GMP (cGMP) content in lecular layer, whereas AMPA raised it in both sections. cerebellar slices prepared from adult rats. The ACPD-in- These results suggest that ACPD enhances the cGMP level duced rise in cGMP level was blocked by compounds through activation of NOS independently of extracellular known to antagonize metabotropic glutamate receptors, calcium, most likely by calcium release from intracellular such as ~~-2-amino-3-phosphonopropio~cacid and L-2- stores triggered by metabotropic glutamate receptors linked amino-4-phosphonobutyric acid, but not by ionotropic glu- to phosphoinositide breakdown, whereas AMPA activates tamate receptor antagonists, D-2-amino-5-phosphonovale- the enzyme by calcium entry from the extracellular space ric acid and 6 - cyano - 7 - nitroquinoxaline - 2 , 3 - dione triggered by ionotropic non-NMDA glutamate receptors. (CNQX), whereas the AMPA-induced rise in cGMP level Key Words: Cyclic GMP-Metabotropic glutamate recep- was suppressed by CNQX. Both rises in cGMP level in- tors-Nitric oxide synthase-Cerebellum-(+)-1-Amino- volved nitric oxide synthase (NOS), because NG-methyl-L- cyclopentane-trans- 1,3-dicarboxylic acid-(R,S)-a- Ami- arginine (NMLA), an inhibitor of NOS, blocked both no - 3 - hydroxy - 5 - methylisoxazole - 4 - propionate. Okada cGMP level rises, and excess L-arginine reversed the effect D. TWOpathways of cyclic GMP production through gluta- of NMLA. After lithium chloride treatment, which could mate receptor-mediated nitric oxide synthesis. J. New+ exhaust phosphatidylinositol phosphates, ACPD no longer chern. 59, 1203-1210 (1992). increased cGMP levels, whereas AMPA was still effective.

It has been shown that glutamate (Garthwaite and phosphoinositide (PI) metabolism exist in abundance Balh, 1978) and its agonists, such as N-methyl+ in the cerebellum (Masu et al., 1991) and can liberate aspartate (NMDA) (Garthwaite et al., 1988)and kain- calcium from intracellular stores (Sugiyama et al., ate (Garthwaite et al., 1989), increase the cyclic GMP 1987). Calcium imaging techniques have revealed (cGMP) content in the cerebellum through activation that calcium level increases occur in the restricted of neural nitric oxide synthase (NOS), a calmodulin- area that depends on their underlying mechanisms. dependent enzyme (Bredt and Snyder, 1990). Be- For example, thc calcium level increase induced by PI cause the effect of these glutamate agonists depends metabolism is restricted to an area near inositol tris- on extracellular calcium (Garthwaite et al., 1988),it is phosphate-releasing sites (Parker and Ivorra, 1990). likely that the Ca2+ required for NOS activation is On the other hand, the calcium level increase due to supplied by calcium influx from the extracellular NMDA receptor activation by presynaptic activity is space during membrane depolarization evoked by restricted to the postsynaptic spines (Mullcr and Con- ionotropic glutamate receptor activation. ner, 199 1). These observations suggest that Ca2+lev- The metabotropic glutamate receptors linked to els increased by different mechanisms have distinct

Received January 28, 1992; accepted March 18, 1992. isoxazole-4-propionate; AP3, 2-amino-3-phosphonopropionic Address correspondence and reprint requests to Dr. D. Okada at acid; AP4, 2-amino-4-phosphonobutyric acid; AP5, 2-amino-5- Laboratory for Neural Networks, Frontier Research Program, RI- phosphonovaleric acid; &MP, cyclic GMP; CNQX, 6cyano-7-ni- KEN,2-1 Hirosawa, Wako, Saitarna 351-01, Japan. troquinoxaline-2,3-dione;ZBMX, 3-isobutyl-1-methylxanthinc; Ahhreviufions used: ACPD, (+)-1-aminocyclopentane-frans-1,3- NMDA, N-methyl-D-aspartate; NMLA, NG-methyl-L-arginine; dicarboxylic acid AMPA, (R,S)-a-amino-3-hydroxy-S-methyl- NOS, nitric oxide synthase; PI, phosphoinositide.

1203 1204 D. OKADA

localizations and hence can play distinct roles (Alkon purchased from Tocris Neuramin (U.K.). (R,S)-a-Amino- and Rasmussen, 1988). Thus, the activation of meta- 3-hydroxy-5-methylisoxazole-4-propionate (AMPA), 2- botropic glutamate receptors is another possible mech- amino-3-phosphonopropionicacid (AP3), and L-2-amino- 4-phosphonobutyric acid (L-AP4) were purchased from anism of NOS activation in the cerebellum. RBI (U.K.). Lithium chloride and EGTA were purchased In the present study, cGMP production mediated from Wako Chemicals and Wako-Dojindo (Japan), respec- by metabotropic glutamate receptor activation was tively. H7 and HA1004 were purchased from Seikagaku investigated in adult rat cerebellar slices. It was found Kogyo (Japan). Other chemicals were purchased from that a selective of metabotropic glutamate Sigma (USA.). receptors, (+)- 1-aminocyclopcntane-trans- 1,3dicar- boxylic acid (ACPD) (Palmer et al., 1989), raised the RESULTS cGMP content through a novel mechanism of NOS activation, which was predominant in the molecular Basal cGMP levels layer and which was independent of extracellular cal- During the preincubation period, the cGMP level cium. In contrast, the AMPA-induced increase in increased transiently (significant at 15-60 rnin), even cGMP content occurred in both the molecular layer though the incubation medium contained neither and the other parts of the cerebellar cortex and de- glutamate agonist nor IBMX (Fig. 1). This basal pended on extracellular calcium. cGMP content then decreased gradually, reaching a stable level: 29.9 _t 2.7 pmol/mg of protein (n = 4) at 90 min, 24.5 f 7.0 pmol/mg (n = 4) at 120 min. The MATERIALS AND METHODS basal cGMP level is higher than that reported by Male Wistar-ST rats (7-1 1 weeks old) were anesthetized Southam and Garthwaite (199 la) (2.94 pmol/mg of with , bathed in ice-cooled water, and decapi- protein), who boiled slices to terminate the reaction, tated. Cerebella were dissected, and parasagittal slices of whereas trichloroacetate was used for termination in vermis (400 pm thick) were prepared at 0°C using a vibrat- the present study. The difference in termination ing slicer (Dosaka, Kyoto, Japan) and transferred to Krebs methods might explain, at least in part, the different buffer (100 ml) at 34°C for preincubation. The buffer con- basal cGMP levels. tained 124 mM NaCI, 5.0 mM KCI, 1.24 mM NaH2P04, The transient increase in basal cGMP level was in- 1.3 mMMgSO,, 2.4 MCaCl,, 26 mMNaHCO,, and 10 mM glucose and was equilibrated with 95% 02/5% C02. hibited by addition of 0.1 mM N"-methybarginhe Usually four to six slices were prepared from one rat. One (NMLA), a specific NOS inhibitor, during preincuba- slice served as the positive control, using 3 mM sodium tion (Fig. 1). Therefore; this increase was due to a nitroprusside to obtain full activation of guanylyl cyclase. spontaneous nitric oxide release, probably evoked by Because the sensitivity of the slices to drugs is highly depen- endogenous agonists that increased intracellular cal- dent on the conditions of preparation, if this control showed cium levels. The measurements of cGMP levels de- a cGMP level rise of <300 prnol/mg of protein (mean _t SE; scribed below were all conducted after the basal 346 t 15 pmol/mg of protein), all the slices were discarded. cGMP level was stabilized by incubation for 90 min. For drug incubation, one slice was transferred to a tube containing 1 ml of Krebs buffer with the drugs at an appro- ACPD-induced rise in cGMP level priate time and kept at 34°C. 0&O2 gas was introduced As shown in Fig. 2A and B, addition of ACPD (at 1 through thin tubes. 3-Isobutyl- 1methylxanthine (IBMX), a mM, for 5 min) raised the cerebellar cGMP content to phosphodiesterase inhibitor, and the agonists were added 59.4 k 2.8 pmol/mg (n = 22), corresponding to 179% after 85 and 90 min of preincubation, respectively. After 5 of the basal level ( p < 0.00 1). The effect of ACPD was min of reaction with the agonist. slices were transferred to glass microhomogenizers and homogenized in 0.2 ml of cold 20% trichloroacetic acid (Wako Chemicals, Japan). The homogenate was centrifuged at 2,000 g for 5 min. The supernatant was washed four times with water-satur- ated diethyl ether and neutralized with a small aliquot of 1 M NaOH to pH 7.4 before determination of cGMP content using a radioimmunoassay kit (Amersham, Japan), in du- plicate. The cGMP content increased linearly with time of agonist exposure, reached a maximum by 3 min, and then did not change with reaction periods up to 10 min (data not

~ shown). The pellet was dissolved in 1 MNaOH, and a small 0 30 60 90 120 aliquot was subjected to protein assay in duplicate by the *at%% method of Lowry et al. (195 1) using bovine serum albumin as the standard. The cGMP content is expressed as pico- FIG. 1. Changes in the basal cGMP level during incubation with- moles per milligram of protein, in mean SE values. Stu- out IBMX at 34°C: control buffer (0) and with 0.1 mM NMLA added at zero-time (A).At zero-time, the slice immediately after dent's t test was performed with Yukms Statistical Library I the preparation at 0°C was transferred to the incubation buffer at software (Yukms Corp.). 34°C. and the slices were incubated further for the periods indi- 6-Cyano-7-nitroquinoxaline-2,3-dione(CNQX), 13-2- cated in the abscissa. Data are mean -c SE (bars) values from the amino-5-phosphonovaleric acid (D-AP5), and ACPD (an indicated number of trials. "p < 0.01 for difference from the 90- equimolar mixture of 1S,3R and lK,3S enantiomers) were min incubation in control buffer.

J. Neurochem.. Vol. 59, No. 4, 1992 cGMP LEVEL RISE BY ME7'ABOTROPIC GLUTAMATE RECEPTOR 1205

A IBMX at 0.1 mM for 10 min, the basal cGMP level 6+ACPD basal I *** was not altered (33.2 f 2.1 pmol/mg, n = 17), whereas at 1 mM, IBMX slightly increased the basal level (43.3 f 2.2 pmol/mg, n = 6; p < 0.05). This dose-response relationship is similar to that of the 30 r IBMX-induced rise in cyclic AMP level in endothelial cells (Hong, 1983). Thus, IBMX at 0.1 mM has vir- tually no effect on the cGMP levels. The cGMP level rise induced by 1 mM ACPD was not observed in the absence of IBMX (25.0 f 2.9 pmol/mg, n = 4), as shown in Fig. 2B. This indicates that cerebellar tissue contains phosphodiesterase activ- ities that prevent the sustained increase of cGMP lev- I els. In all experiments except for those shown in Figs. :y 0.1 0.3 0 0.1 0 0.1 1.0 1,2B, and 2C, the reaction mixture contained IBMX FIG. 2. A The ordinate expresses the cGMP level increase at 0.1 mM. above the basal level, in picomoles per milligram protein per 5 rnin, rise in response to ACPD. "p< 0.01, "'p < 0.001 for difference from AMPA-induced in cGMP level basal level. 6:Effect of IBMX on the ACPD (1 mM)-induced rise in As shown in Fig. 4, AMPA at concentrations of cGMP level. ***p < 0.001 for difference from the corresponding >0.3 pM also induced a cGMP level rise in the adult basal value shown in C (0.1 rnM IBMX). C: Effect of IBMX on rat cerebellum in a dose-dependent manner. In con- basal cGMP levels. *p < 0.05 for difference from IBMX at 0 mM. trast to ACPD, the cGMP level rise induced by 0.3 or Data are mean +- SE (bars) values. I pM AMPA was effectively suppressed by 0.1 mM CNQX: 0.3 pMAMPA plus CNQX, 28.3 ? 2.5 pmol/ mg, n = 5. Therefore, the effect of AMPA was due to dose dependent, as shown in Fig. 2A. ACPD at 0.3 activation of non-NMDA ionotropic glutamate re- mM was as effective as at 1 mM(n = 6, p < 0.0 I), but ceptors. The AMPA-induced rise in cGMP levels also its effect was not significant at 0.1 mM (n = 8) (see required 0.1 mM IBMX (data not shown). When also Southam and Garthwaite, 199 lb). This dose-re- AMPA and ACPD were applied simultaneously, sponse relationship was similar to that of the ACPD- there was no indication of any associative effects (data induced C1- current obtained from Xenopus oocytes not shown). expressing metabotropic glutamate receptors by in- jection of rat brain RNAs (Tanabe et al., 1991). Involvement of nitric oxide Ionotropic glutamate receptor antagonists did not Because agonist-induced rises in cGMP levels re- quired the coexistence of low concentrations of affect the cGMP level rise induced by 0.3 mM ACPD IBMX, it is necessary to determine which process was (0.2 mM D-AP~,57.5 +- 4.3 pmol/mg, n = 5; 0.1 mM rise: CNQX, 51.3 f 1.9 pmol/mg, n = 4), nor did they responsible for the cGMP level inhibition of affect the basal cGMP levels (D-AP~,30.6 k 5.5 phosphodiesterase or activation of guanylyl cyclase. pmol/mg, n = 4; CNQX, 34.3 * 0.5 pmol/mg, n = 4). These results (Fig. 3) show that the ionotropic gluta- mate receptors were not involved in the ACPD-in- 1 ***- *** duced rise in cGMP level. Antagonistic effects of L-AP~(Nicoletti et al., 1986) and DL-AP~(Schuepp and Johnson, 1989) on the PI breakdown induced by metabotropic glutamate re- ceptors have been reported. In contrast to DAP~and CNQX, 1 mM L-AP~and AP3 significantly (p < 0.001) reduced the cGMP level rise induced by 0.3 mM ACPD to 32.5 -t 6.1 and 43.5 * 3.7 pmol/mg (n = 6), respectively, as shown in Fig. 3. They did not alter the basal cGMP levels: L-AP~,29.1 f 7.3 pmol/ mg, n = 6; AP3, 35.5 +- 2.7 pmol/mg, n = 4. These pharmacological properties resemble those of meta- crmx D-Aps AP3 L-AP4 botropic glutamate receptors and strongly suggest that ACPD raises cGMP levels in adult rat cerebellar slices FIG. 3. Effect of glutamate receptor antagonists on the rise in cGMP levels induced by 0.3 mM ACPD: no antagonist, 0.1 mM through metabotropic glutamate receptors. CNQX, 0.2 rnM D-AP~,1 mM AP3, and 1 mM L-AP~.Dotted Effect of IBMX columns represent ACPD-induced cGMP levels. Data are mean f SE (bars) values. ***p < 0.001 for difference from correspond- Figure 2C shows the effects of IBMX on the basal ing basal levels (without ACPD but with antagonist; open cd- cGMP levels. When the slices were incubated with urnns).

J. Neurochem.. Vol. 59. No. 4, 1992 1206 D. OKADA

1982; Worley et al., 1988). The basal cGMP level is 2 enhanced by spontaneous NOS activity during the preincubation period, as shown in Fig. 1, and this NOS activity should be evoked by endogenous ago- nists, such as glutamate. It is also well known that the cerebellum is enriched with PI metabolism triggered by glutamate (Hwang et al., 1990). Hence, it is likely that this transient increase in the basal cGMP level is accompanied by spontaneous PI turnover. Thus, it is expected that the lithium chloride treatment during the preincubation period exhausts the resource of ino- sitol 1,4,5-trisphosphate and subsequently suppresses 5 the action of ACPD on PI turnover. Lithium chloride at 3 mM was added to the prein- 0 1 cubation medium 1 h before addition of 1 mM h- h- ACPD. As shown in Fig. 6, lithium chloride signifi- 0 0.1 0.3 1 10 3 30 100 cantly suppressed the effect of ACPD (31.7 2.8 =(MM) * pmol/mg, n = 7; p < 0.001). The lithium treatment FIG. 4. Dose-response relationshipof AMPAinduced cGMP pro- did not alter the basal level (25.6 5 3.4 pmol/mg, duction (0) and effect of 0.1 mM CNQX on the AMPA-induced n = 5). In contrast, the AMPA-induced rise in cGMF rise in cGMP ieveis (V). Data are mean & SE (bars) values irom the indicated number of trials. “‘p < 0.001 for difference from the level was not affected by the same treatment (65.6 corresponding controls (without CNQX). +. 2.2 pmol/mg, n = 4; Fig. 6B). Thesc results support the hypothesis that an ACPD-induced rise in cGMP level involves PI breakdown, whereas the basal cGMP level and AMPA-induced rise in cGMP level are inde- When the slices were incubated with 0.1 mM NMLA pendent of PI breakdown. (plus 0.1 mM IBMX), a competitive inhibitor of NOS, for 10 min before addition of ACPD, the effect of ACPD was completely blocked (Fig. 5; 17.2 k 6.0 Effect of calcium depletion Metabotropic glutamate receptors may increase in- pmol/mg, n = 5;p < 0.0 1), and the basal level was not tracellular calcium levels through a pathway other changed (24.9 * 5.5 pmol/mg, n = 5). The effect of NMLA was antagonized by 1 mM L-arginine, which than PI-induced liberation of calcium from intracel- is the physiological substrate for NOS, as shown in lular stores because this receptor activates protein ki- nase C, which closes potassium channels (Baraban et Fig. 5 (76.3 & 8.8 pmol/mg, n = 4; difference from NMLA plus ACPD was significant at p < 0.001). al., 1985); the subsequently induced depolarization NMLA plus L-arginine did not alter the basal level (39.4 k 4.1 pmol/mg, n = 4). These results indicated that ACPD raised the cGMP content through activa- I** tion of NOS. Bo c ACPD T The cGMP content rise induced by 0.3 pLM AMPA .....-...-.- ***- was also dependent on NOS. It was inhibited by ...-.- NMLA (1 3.1 k 2.2 pmol/mg, n = 5), and L-arginine . antagonized the effect of NMLA (56.6 f 3.5 pmol/ mg, n = 5). Thus, both metabotropic and ionotropic %., I glutamate receptor agonists activate NOS to increase .P the cGMP content in rat cerebellar slices. NOS pro- duces nitric oxide, which activates guanylyl cyclase. Therefore, it is likely that the agonist-induced rises in ii cGMP levels are evoked through activation of gua- - nylyl cyclase. 20 ...:.:2...... Involvement of PI metabolism fl .. Metabotropic glutamate receptors linked to PI me- 0- .. tabolism liberate calcium from intracellular stores mtml NMLA NMLA control MA NMLA (Sugiyama et al., 1987). The Ca2+required for ACPD- L-ARG L-ARG induced NOS activation can thus be supplied by this FIG. 5. Effects of NMLA (0.1 mM) and L-arginine (L-ARG; 1 mM) mechanism. It is well known that lithium chloride on the rise in cGMP level induced by 1 mM ACPD(B) and 0.3 pA4 inhibits inositol formation, and when PI turnover is AMPA (Ed). Basal cGMP levels are also shown (0). The “control” data are identical with those in Figs. 26 and 4. Data are mean active, this inhibition subsequently inhibits the repro- ? SE (bats) values. “’p < 0.001 for difference between basal and duction of phosphatidylinositols (Bemdge et al., agonist-induced cGMP levels.

J. Neurochem.. Vol. 59, No. 4. 1992 cGMP LEVEL RISE BY METABOTROPIC GLUTAMATE RECEPTOR 1207

A protein in each region and was compared with the **t entire slice measurement. These values (entire slice *f' observed, 33.2 * 2.1 pmol/mg, n = 17; calculated, ...... 38.3 2.4 pmol/mg, n = 3) were similar, and the ... difference was not significant,suggesting that the enzy- matic activities were terminated by the microwave irradiation and that there was no loss of cGMP after separation of the layers. ACPD raised the cGMP content mainly in the mo- .. lecular layer. The increase in cGMP level above the [ basal value in the molecular layer (obtained from the .... average of the ACPD-induced level minus that of the .... basal level in each part) was 55.5 4 11.7 pmol/mg (n = 3), significantly larger than the 7.5 f 4.4 pmol/mg control LEI control LiCl (n = 3) of the remainder (p < 0.05). The calculated FIG. 6. Effect of incubation with 3 mM LiCl for 1 h on (A) 1 mM content of the entire slices matched the observed val- ACPD- and (B) 0.3 pM AMPA-induced rises in cGMP levels. Basal ues: observed, 59.4 f 2.8 pmol/mg, n = 22; calcu- cGMP levels are also shown (0).The data without LiCl are from lated, 59.0 k 2.6 pmol/mg, n = 3. Figs. 2B and 4. Data are mean f SE (bars) values. "*p < 0.001 AMPA raised the cGMP content of both the molec- for difference between agonist-induced and basal cGMP levels. ular layer and the remaining part of the cerebellar slices. The increases above the basal level in the molec- ular layer and in the remainder were 64.0 13.1 and would enhance calcium influx through voltage-gated k 35.0 -t 18.4 pmol/mg (n = 5), respectively. Both ago- calcium channels. To test this possibility, an experi- nists raised the cGMP content similarly in the molecu- ment was conducted using an extracellular medium lar layer, whereas AMPA induced a larger rise than depleted of Ca2+. ACPD in the remainder. The calculated and observed The application of 1 mM ACPD to slices preincu- contents of the entire slice were 77.2 6.6 (n = 5)and bated in a calcium-free medium (0.2 mM EGTA * 88.5 4.4 (n = 7) pmol/mg, respectively. without CaCl, for 15 min) raised the cGMP content * to levels similar to those in the normal calcium me- DISCUSSION dium (63.5 -t 6.4 pmol/mg, n = 5), as shown in Fig. 7. The difference from the normal condition was insig- Because no specific antagonist for the metabotropic nificant. This result showed that the ACPD-induced glutamate receptor is available, various receptor antag- rise in cGMP levels was independent of extracellular onists were tested against the most specific agonist, calcium and suggested the involvement of calcium ACPD (Palmer et al., 1989), to evaluate the involve- release from intracellular stores. ment of this receptor. In addition, the involvement of In contrast, under the same calcium-depleted con- dition, the effect of AMPA at 0.3 pLM was completely = suppressed (Fig. 7; 24.5 -+ 2.8 pmol/mg, n 4), and ACPD AMPA the difference from the normai condition was signifi- **t *t* cant (p< 0.001). This result indicates that, unlike the T ACPD-induced rise in cGMP levels, the AMPA-in- duced rise depends on calcium influx from the extra- cellular space. Layer analysis In some experiments, the slices were put on glass slides immediately after reaction and fixed by micro- wave irradiation for 15 s (RE-M 15 microwave oven, 500 W, 2.45 GHz; Sharp Co. Ltd., Japan). The molec- ular layer (including the Purkinje cell layer) was then isolated, and the cGMP content was measured sepa- rately for the molecular layer and the remainder (the granule cell layer plus white matter). The results are +ca Ca +Ca Ca shown in Fig. 8. FIG. 7. Effect of calcium depletion on 1 mM ACPD- and 0.3 pM In control slices (without agonists), the two parts AMPA-induced rises in cGMP levels. Normal medium (denoted as showed similar basal cGMP contents: molecular +Ca) contained 2.4 mM CaCI,, and calcium-depleted medium (de- noted as -Ca) contained 0.2 mM EGTA without added CaCI,. layer, 33.5 8.3 pmol/mg; remainder, 42.5 k 11.5 2 Basal cGMP levels are also shown (0).Data are mean f SE (bars) pmol/mg (n = 3). The cGMP content of the entire values. "*p < 0.001 for difference between agonist-induced and slice was calculated from the amount of cGMP and basal cGMP levels.

J. Neurochem.. Vol. 59, No. 4. 1992 1208 D. OKADA

g+wm 2C). The agonist-induced rises in cGMP level are due to NOS activation but not to phosphodiesterasc inhi- bition because the agonist-induced cGMP rise is inhib- ited by NMLA, an NOS inhibitor (Fig. 5). Second, T Hong (1983) reported the effect of IBMX on phos- pholipases, which was obvious at concentrations of > I mM, suggesting that 0.1 mM IBMX does not af- fect phospholipases. Third, Breer et al. (1 990) showed rapid formation of cyclic AMP and inositol trisphos- phates in olfactory transduction. These second mes- sengers were degraded within some 100 ms. It is likely that the phosphodiesterase in the cerebellum also de- grades cGMP produced in response to ACPD or AMPA very rapidly. The involvement of PI breakdown in ACPD re- sponses was indicated by blockade with lithium chlo- ride and also through the dependence on extracellular calcium. The results of these experiments clearly dis- criminated the effects of ACPD and AMPA. The ACPD-induced rise in cGMP !evels wi?s suppressed by lithium chloride, but the AMPA-induced rise was ACPD AM not. From layer analysis, the ACPD-induced rise in basal cGMP level was observed mainly in the molecular FIG. 8. Layer analysis in the molecular layer (mol) and the remaln- layer. This distribution is consistent with autoradio- ing part [granular cell layer plus the white matter (gr + wm)]. Basal graphic demonstration of ACPD-induced PI turnover cGMP levels are indicated (0)below the ACPD (a)-and AMPA (Hwang et al., 1990). The ACPD-induced rise in (&induced increase in cGMP levels above the basal level. The cGMP level was not altered by incubation with 0.2 ordinate shows the total cGMP levels. Data are mean 2 SE (bars) values. One slice contained 1.3-2.0 (mean, 1.65) mg of protein. mM EDTA medium, whercas the AMPA-induced The molecular layer from one slice contained 0.32 mg of protein rise was completely reduced, showing that under the and 1.07 mg for the remainder (average protein yield, 84%). present conditions, calcium depletion did not affect 'p < 0.05 for difference between ACPD-induced increases in the intracellular calcium stores but effectively re- cGMP levels in the molecular layer and the remainder. duced extracellular calcium activity. More vigorous conditions to deplete extracellular calcium will ex- haust intracellular stores, and, in fact, incubation with PI breakdown and the independence of extracellular a medium containing 0.3 mM EGTA for 20 min de- calcium were studied. From this combination of ex- creased the ACPD-induced rise in cGMP levels (data periments, the present results show that NOS is acti- not shown). These observations suggest that in the vated by thc metabotropic action of glutamate recep- ACPD-induced rise in cGMP levels, NOS is activated tors, triggered by ACPD, in a manner independent of by intracellular calcium release. extracellular calcium, most likely due to intracellular The involvement of protein kinase C, whose activa- calcium liberation. Nitric oxide thus produced acti- tion leads to calcium entry (Baraban et al., 1985), is vates guanylyl cyclase and thereby enhances the excluded from the ACPD-induced rise in cGMP lev- cGMP content in the molecular layer. AMPA also els, which occurs under calcium-free conditions. How- raised the cGMP content in the cerebellum through ever, protein kinases can be involved in an ACPD-in- activation of NOS, but this effect was dependent on duced rise in cGMP levels as long as their action or extracellular calcium, suggesting the involvement of activation is independent of calcium entry. In fact, calcium entry. protein kinase inhibitors (staurosporine at 0.5 pM for The basal cGMP lcvel after 90 min of preincuba- 15 min, H7 at 0.3 mM for 60 min, HA1004 at 0.3 tion is maintained, at least in part, by an equilibrium mM for 60 min, and polymyxin B at 0.1 mM for 60 between NOS and phosphodiesterase activities, as rnin), as well as a protein kinase C activator (phorbol judged from the effects of IBMX and NMLA (Figs. 2 12,13-diacetate at 0.5 pM for 10 min), enhanced the and 5). The basal cGMP level might be maintained basal cerebellar cGMP levels (author's unpublished also by NMDA receptor activation, as Southam and data). These results suggest the interesting possibility Garthwaite (19914 reported. In the present study, that protein kinases can regulate the cerebellar cGMP however, D-AP~did not significantly alter the basal level through modification of NOS, because the level (Fig. 3). Both AMPA- and ACPD-induced rises cloned NOS has putative phosphorylation sites (Bredt in cGMP levels required 0.1 MIBMX.This require- eta]., 1991). ment is well explained by the effect of IBMX as a Thus, NOS is activated through two different path- phosphodicsterase inhibitor. First, 0.1 mM IBMX ways in cerebellar slices. One pathway is mediated by does not significantly affect the cGMP level itself (Fig. ionotropic glutamate receptors and depcnds on extra-

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