3 s) and after predesensitization of GABAA recep- tors. Baclofen also inhibited. '6Cl- flux by cortical mem- branes but was more effective with cerebellar prepara-.
Cerebellar
GABAB receptors
modulate
function
LISA HAHNER, SUSAN MCQUILKIN,1 AND R. ADRON HARRIS*,t,’ *I/etera Administration Medical Center, and Department of Pharmacology, University Colorado 80262,
Interactions between GrABAA and GABAB receptors were studied using muscimol-stimulated uptake of 36C1 by membrane vesicles from mouse cerebellum. Baclofen inhibited muscimol-stimulated 36Cl uptake and this action was more pronounced with longer flux times (30 vs. 3 s) and after predesensitization of GABAA receptors. Baclofen also inhibited ‘6Cl- flux by cortical membranes but was more effective with cerebellar preparations. The action of baclofen was stereoselective, calcium-dependent, and blocked by the GABAB receptor antagonist 2-OH-saclofen. It was mimicked by GTP-y-S but not by GDP--S, which suggests that baclofen may be acting via a G protein. The action of baclofen was inhibited by U73122, an inhibitor of phospholipase C. However, the potassium channel blockers tetraethylammonium or Ba2’ did not affect the action of baclofen. The results show that activation of GABAB receptors can inhibit the function of GABAA receptors and suggest that this action involves either a nondesensitizing subtype of GABAA receptor or the rate of recycling of desensitized to nondesensitized receptors. We speculate that this action of baclofen results from activation of phospholipase C and phosphorylation of a subtype of GABAA receptor by protein kinase C.-Hahner, L.; McQuilkin, S.; Harris, R. A. Cerebdllar GABAB receptors modulate function of GABAA receptors. FASEBJ. 5: 2466-2472; 1991. Key Words: GA BA A receptors . GA BA B receptors nels cerebellum . guanine nucleotides . protein lion protein kinose C phospholipase C
chloride chanphosphoryla-
MAJOR INH!B1mRY NEUROTRANSMITTER in the mammalian brain is ‘y-aminobutyric acid (GABA),2 which is known to act through two subtypes of receptors, GABAA and GABAB. The GABAA receptors are integral components of a chloride channel complex, whereas GABAB receptors are coupled to potassium and calcium channels as well as other secondmessenger systems by guanine nucleotide-binding (G) proteins (1). Baclofen is the prototypical GABAB agonist and muscimol is the most commonly used GABAA agonist. The two receptors are colocalized on cells in the dorsolateral septal nucleus (2),on dorsal root ganglion primary afferents (3, 4), on adrenal chromaffin cells (5), and in the CAl region of hippocampus (6). Colocalization raises the possibility of interactions between the two receptors; specifically, the neurotransmitter actions of GABA at the GABAA receptor might be modulated by actions of GABA at GABAB receptors. However, we are aware of only two reports of baclofenmediated changes in chloride channel function; in one, baclofen enhanced 35Cl- uptake by crayfish muscle (7); in the other, baclofen inhibited muscimol-stimulated 36Cl uptake by cultured cerebellar granule cells (8). However, neither study presented detailed evidence that the observed actions of baclofen resulted from activation of GABAB receptors. The binding of baclofen to GABAB receptors requires calcium, and effects of baclofen are transduced by G proteins.
2466
of Colorado
Health
Sciences
Center, Denver,
USA
ABSTRACT
THE
of GABAA receptors
Thus, actions of baclofen at GABAB receptors should be concentration-dependent, stereoselective, dependent on calcium, mediated by a G protein, and antagonized by 2-OHsaclofen (1, 9). The purpose of the present study was to use these criteria to determine if activation of GABAB receptors modifies GABAA action. In addition, we asked the following questions: Can cerebellar or cortical membrane vesicles be used rather than cultured granule cells to study the interaction? Can the action of baclofen be altered by inhibitors of potassium channels or phospholipase C (i.e., possible effects of GABAB receptor activation)? Last, many GABAAactivated chloride channels desensitize on a millisecond time scale (10) but Kardos (8) used uptake times of 7-14 s to study interactions of baclofen and muscimol. This raises the possibility that Kardos studied a nondesensitizing (or slowly desensitizing) type of GABAA receptor that is affected by baclofen. METHODS Materials Drugs and chemicals used and their respective sources were as follows: Muscimol, picrotoxin, guanosine 5-0(3-thiotriphospate) (GTP-’y-S), guanosine 5’-O(2-thiodiphosphate and (GDP-/3-S), Sigma Chemical Company (St. Louis, Mo.); 2-OH-saclofen and bicuculline methiodide, Research Biochemicals Incorporated (Natick, Mass.); U73122,
The
Upjohn
Company,
(Kalamazoo,
Mich.);
+,
-,
and +1baclofen, Ciba-Geigy (Summit, NJ.), and 36cl (specific activity 6.5 mCi/g Cl), ICN (Irvine, Calif.). All other reagents used were of analytical grade and were purchased from Sigma Chemical Company or Fisher Scientific (St. Louis, Mo.). Membrane
preparation
Adult (60-90 days) male ICR mice were obtained from Harlan Sprague Dawley, Incorporated (Indianapolis, Ind.). The procedure for preparation of membrane vesicles (microsacs) has been described by Harris and Allan (11). Mice were killed by decapitation, and the cerebellum or cerebral cortex was dissected and retained. For each preparation, 4-10 cerebella or 1-3 cortices were pooled. Tissue was homogenized by hand (10-12 strokes)in 4.5 ml of ice-coldassay buffer [145 mM NaC1, 5 mM KC1, 1 mM MgC12, 10 mM
‘To who all correspondence should be addressed, at: Department of Pharmacology, Campus Box C-236, University of Colorado Health Sciences Center, 4200 East Ninth Ave., Denver, CO 80262, USA. 2Abbreviations: DMSO, dimethylsulfoxide; GABA, ‘y.aminobutyric acid; GDP-I-S, guanosine 5’-O-(2-thiodiphosphate); GTP-7-S, guanosine 5’-O-(S-thiotriphosphate); HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid; PKC, protein kinase C; TEA, tetraethylammonium; U73122, 1-[6-[[17fl-3-methoxyestra-l,3,5(10)trien-17-yl]amino]hexyl]-IH-pyrolle-2,5-dione; G protein, guanine nucleotide-binding protein; ANOVA, analysis of variance. 0892-6638/91/0005-2466/$01
.50. © FASEB
D-glucose, 1 mM CaCl2, and 10 mM 4-(2-hydroxyethyl)-1piperazonethanesulfonic acid adjusted to pH 7.5 with Tris base] using a glass Teflon homogenizer (Thomas, size C). The homogenate was centrifuged at 900 x g for 15 mm. The supernatant was decanted and the pellet was resuspended in 8 ml of assay buffer and centrifuged at 900 x g for 15 mm. The final pellet was suspended in assay buffer, yielding a preparation containing 4-5 mg protein per milliliter of suspension. Microsac membranes were lysed and resealed as described previously (12). In brief, microsacs were prepared as described previously until the second resuspension, when the pellet was resuspended in lysing buffer (6 mM tricineand 1 mM Mg acetate adjusted to pH 7.5 with Tris base). Guanosine 5’-O-(3-thiotriphosphate (100 sM) or GDP-f3-S (100 tM) was then added to the samples followed by homogenization. These preparations were incubated at 34#{176}C for 10 mm and resealed with 10 volumes calcium-free buffer [145 mM NaCl, 5 mM KC1, 1 mM MgC12, 10 mM D-glucose, 10 mM 4-(2-hydroxyethyl)-piperazinethanesulfonic acid, 3 mM ethyleneglycol-bis-(f3-aminoethyl ether) N,N,N’N’-tetraacetic acid adjusted to pH 7.5 with Tris base]. The resealing buffer was gradually added over 5 mm while membranes remained at 34#{176}C. After the final addition of resealing buffer, membranes were kept at 34#{176}C for 5 mm and then placed on ice for 5 mm. Resealed membranes were centrifuged at 17300 x g for 5 mm, the supernatant was decanted, and the pellet was resuspended in assay buffer yielding a preparation containing 4-5 mg protein/ml suspension. Procedure
for 36C1- uptake
This procedure was modified from that of Allan and Harris (13). Microsacs (200 1d) were incubated in a 34#{176}C water bath for 5 mm. Uptake was then initiated by addition and immediate vortexing for 200 l of a solution maintained at 34#{176}C containing 36Cl (2 uCi/ml of assay buffer) with or without 3 tM muscimol and with or without baclofen. Other drugs (2-OH saclofen, U73122, TEA, BaC12, or bicuculline methiodide) were added to the membranes at the beginning of the 5-mm incubation and to the 36Cl solution. In some experiments muscimol was also added prior to the incubation, and baclofen was added 30 s before addition of 36C1. Thirty seconds (unless indicated otherwise) after addition of 36Cl, influx was terminated by 4 ml ice-cold assay buffer containing 100 ,tM picrotoxin, and rapid filtration under vacuum (six in Hg) Onto a premoistened GB100R filter (MicroFiltration Systems, Dublin, Calif.), using a Hoefer manifold (Hoefer Scientific, San Francisco, Calif.). These filters were washed with an additional 12 ml ice-cold assay buffer containixTg 100 tM picrotoxin with the manifold towers removed. The amount of radioactivity on the filters was determined by liquid scintillation spectrometry. The amount of 36Cl bound to the filters in the absence of membranes (no tissue blank) was substracted from all values. Muscimol-dependent uptake (i.e., influx through GABAA receptor-activated Cl channels) was defined as the amount of 36C1 taken up while muscimol was present in the medium (total uptake) minus the amount of 36C1 taken up when muscimol was not present (basal or muscimol-independent). When evaluating the effectsof baclofen, 2-OH saclofen,or U73122 on the muscimol-dependent uptake, basal uptake was also measured in the presence of these drugs and the values were used to determine the muscimol-dependent uptake for that drug. All drugs were dissolved in water except for U73122, which was dissolved in dimethyl sulfoxide (DMSO); the final concentration of DMSO in the assay was
GABAA AND
GABAB RECEPTOR INTERACTIONS
0.1%
and
was
tested
as a vehicle
control
for
allexperiments
with U73122. Statistics An analysis of variance (ANOVA) for repeated measures was used to compare time course data and concentration response curves. A Scheffe test was used posthoc with ANOVAs. For experiments involving only two groups, a Student’s t test was used.
RESULTS Cerebellar membrane vesicles (microsacs) were chosen for initial studies. The time course of muscimol-stimulated 36C1 uptake consisted of an initial rapid phase of uptake into these vesicles that was complete by 10 s, followed by a slower phase that continued for at least 60 s (Fig. 1, upper and middle panels; 60 s not shown). Without preincubation, baclofen did not alter uptake produced by a 3- or 10-s exposure to muscimol, but significantly inhibited uptake at the 30- or 60-s time periods (Fig. 1, upper panel; 60 s not shown). There are two possible explanations for this result. Because baclofen was added with the muscimol and 36Cl, the membranes were exposed to baclofen for 3-60 5; thus baclofen may have a slow onset of action and its inhibitory action is observed with a 30- to 60-s exposure. Alternatively,exposure to muscimol for even a few seconds produces desensitization of GABA-activated chloride channels (10), and baclofen may inhibit only the nondesensitizing channels that contribute to the 36Cr uptake mainly at 30- to 60-s uptake times. To test these possibilities, membranes were preincubated with baclofen (200 tiM) for 30 s before initiatinguptake. In addition, membranes were preincubated with muscimol (3 tM) for 5 mm before exposure to 36Cl to desensitize some of the channels (10, 14). Preincubation with baclofen produced some inhibition of muscimol-stimulated 36Cl uptake at 3 or 10 s (Fig. 1, middle panel); however, predesensitization with muscimol followed by 3 s exposure to baclofen did not inhibit uptake (data not shown). Muscimol predesensitization combined with 30 s preincubation with baclofen resulted in clear inhibition of uptake at 3 or 10 s (Fig. 1, lower panel). The initial, rapid phase of 6Cl uptake was greatly reduced by predesensitization (Fig. 1; compare lower panel with middle and upper panels). Also, the inhibition produced by baclofen with 30 s flux time is remarkably consistent (32-34%, see legend to Fig. 1) regardless of preincubation conditions. Thus, it appears that the action of baclofen is time-dependent, requiring about 30 s, and that it primarily inhibits a slow or nondesensitizing channel. The 36C1 uptake produced by 30 s exposure to muscimol was blocked by the GABAA receptor antagonist, bicuculline methiodide (control uptake was 34 ± I nmol/mg protein, uptake with 100 sM bicuculline was 6 ± 2 nmol/mg protein, n=3). This indicates that the uptake of 36Cl is mediated by a GABAA receptor. For subsequent experiments, a 30-s uptake time was used without muscimol predesensitization and without preincubation with baclofen. Baclofen did not inhibit the muscimol-independent (basal) uptake of 36c1. A 30-s exposure to 36cl resulted in chloride accumulation of 87 ± 3 for control and 87 ± 4 for the baclofen (300 tM) treatment (units are nmol Cl/mg protein; mean ± SEM, n=6). Next we tested the dependence of the baclofen inhibition on muscimol concentration. The muscimol EC50 was about 500 nM in the presence or absence of baclofen, but the up-
2467
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1. Time course of muscimol-stimulated uptake of 36C1- by cerebellar microsacs and inhibition of uptake by baclofen. The concentration of muscimol was 3 sM, the concentration of (± )baclofen was 200 M. Upper panel) Muscimol, baclofen, and 36C1- were added at zero time. Percent inhibition by baclofen: 3 s, 4%; lOs, 10%; 30 s, 34%. Values are mean ± SEM, n=3. Significant effect of baclofen, P < 0.005, ANOVA. Middle panel) Muscimol and 3tClwere added at zero time and baclofen was added 30 s before zero time. Percent inhibition by baclofen: 3 s, 18%; 10 s, 21%; 30 s, 34%. Values are mean ± SEM, n=4. Significant effect of baclofen, P < 0.02, ANOVA. Lower panel) Muscimol was added 5 mm be-
fore zero time to produce
receptor
desensitization,
baclofen
In some cases the symbols
and
about microsacs,
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half
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as all
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as studies
that used
this
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Vol. 5
July 1991
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and GDP-i3-S, which inhibits G protein activation-into the membrane vesicles. Based on other studies (see Discussion), GTP--y-S but not GDP-j3-S would be expected to mimic the action of baclofen. Lysed/resealed vesicles showed a muscimol-dependent uptake of 36C1-, although the uptake was less than that obtained from intact microsacs (Fig. 4 vs. Fig. 1). Baclofen did not inhibit this uptake of 36C1- by lysed/resealed microsacs. However, microsacs resealed with GTP-’y-S
with
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2468
C
are larger than the error bars.
take produced by a maximal concentration of muscimol was reduced by baclofen (Fig. 2). A muscimol concentration of 3 M was chosen for all further experiments. This concentration usually produced an uptake of 30-40 nmol.mg’.protein’.30 s, although larger uptakes (50-60 nmol) were seen in some experiments. The basis of this variation is not known. The concentration dependence and stereoselectivity of the inhibitory action of baclofen was also examined. The (+) isomer of baclofen was inactive, but the (-) isomer and racemic mixture each inhibited muscimolstimulated 36C1- uptake by 30-40% (Fig. 3). The IC50 was about 50 ILM for the (-) isomer and about 100 tM for the racemic mixture, indicating that the (+) isomer had neither agonist nor antagonist actions in this system. We carried out our assays at a temperature of 34#{176}C, but Kardos (8) used 0#{176}C to study interactions of GABAA and GABAB receptors. We repeated some of our experiments at 0#{176}C and found that actions of baclofen were similar at 0 and 34#{176}C (300 M baclofen inhibited uptake by 39 ± 7%, n=8, at 0#{176}C). Microsacs prepared from mouse cortex were also tested and found to display muscimol-dependent uptake of 36Cl- similar to that of cerebellar microsacs (38 ± 2 nmol Cl/mg protein’.30 s’ with 3 M muscimol), and baclofen concentrations of 60, 200, or 600 ILM inhibited uptake by 8, 20,
o CONTROL
40
=
was ad-
ded 30 s before zero time, and 36Cl- was added at zero time. Percent inhibition by baclofen: 3 s, 26%; 10 s, 40%; 30 s, 32%. Values are mean ± SEM, n.4. Significant effect of baclofen, P < 0.05,
ANOVA.
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a-
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2. Concentration
dependence
of muscimol-stimulated
in the presence and absence of are mean ± SEM, n3. In some than the error bars.
The FASEB Journal
(±)baclofen cases the
36C1
(200
,uM).
symbols
are
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ET AL.
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as a percentage
of that
obtained in the absence of baclofen (control uptake). Muscimol concentration was 3 M, uptake time was 30 s. Values are mean ± SEM, n 3. In some cases the symbols are larger than the error bars. baclofen produced a small (but statistically insignificant) further inhibition of uptake in the microsacs resealed with GTP-’y-S (Fig. 4). Inclusion of GDP-fl-S during lysing/resealing did not alter 36Cl uptake (Fig. 4). The GABAB receptor antagonist 2-OH-saclofen antagonized the action of baclofen, but interpretation of this effect of saclofen is complicated by the observation that 2-OH-saclofen also inhibited the muscimol-dependent uptake of 36Cl (Table 1). The action of baclofen was dependent on calcium as baclofen failed to inhibit the muscimolstimulated uptake of 36Cr in the absence of calcium (Table 1). Removal of calcium did not affect 56Cl- uptake (data not shown), suggesting that the membrane integrity was not compromised by the calcium-free solution,but toxic effects of this manipulation cannot be ruled out. The potassium channel blockers Ba2’ and TEA had no effect on the action of baclofen (Table 2). However, Ba2’ enhanced 36Cl- uptake, making it difficult to interpret results obtained with baclofen and Ba24. The compound U73122 blocked the inhibitory effectof baclofen at concentrations (0.1-I tiM) where it acts as a specific inhibitor of phospholipase C (15). U73122 itself produced a slight inhibition of muscimol-stimulated 36cl uptake (Fig. 5).
Control
Molecular cloning (16) and receptor binding experiments utilizingmultiple ligands and autoradiographic analysis(17) suggest the existence of many differentsubtypes of GABAA receptors in mammalian brain. Binding of GABAA agonists to brain membranes distinguish at least three sites with affinities of approximately 5-10 nM, 100-500 nM, and 10-100 eM (18, 19). Functional studies indicate that the lower-affinity sites are responsible for opening the chloride channels, which may be further resolved into three different types based on channel kinetics. One receptor desensitizes in about 200 ms, another in several seconds, but both have an affinity for GABA of about 100 sM (14). A third, nondesensitizing receptor has been noted in several electrophysiological studies (20, 21).Application of muscimol to brain mem-
GABAA AND GABA5 RECEPTOR INTERACTIONS
GOP$S100pM
Figure 4. Effects of baclofen and GTP-y-S on the muscimolstimulated uptake of 36C1 by cerebellar microsacs. Vesicles were lysed and resealed in the absence or presence of 100 CM GTP-’y-S and the effects of(±)baclofen were determined on 36Cl- flux. Muscimol concentration was 3 ,LM, uptake time mean ± SEM, n=8. Significantly different P < 0.02; S* < 0.01 (1 test).
was 30 s. Values are from control values,
brane vesicles for 3 s stimulated uptake of 36Cl- primarily through the two rapidly desensitizingchannels, and this action of muscimol was reduced by baclofen by only 18% in the present studies. However, 36C1- uptake resulting from prolonged (30-60 s) exposure to muscimol was inhibited by 35-40% by baclofen. Two possible explanations are that baclofen enhanced effluxof 36C1 by stimulating a chloride pump or exchanger or that it inhibited uptake through channels. The first hypothesis is not supported by the observation that although the muscimol-independent accumulation of 36Cl was substantial after 30 s, baclofen did not reduce this basal uptake of 36C1, indicating that baclofen inhibited uptake through a channel. Information about this channel is provided by the observation that predesensitization of GABAA receptors by preincubation with muscimol revealed a baclofen inhibition of 26-40% at uptake times of 3-10 s, suggesting that the remaining, nondesensitizing receptors were affected by baclofen. The pharmacology of the uptake produced by 30 s exposure to muscimol is somewhat different from that TABLE 1. Antagonism of the inhibitory action of baclofrn on muscimolstimulated chloride uptake by saclofen or calcium-free buffer Treatments
DISCUSSION
GTP#{212}S 100pM
Control Baclofen, 100 tM Saclofen, 30 eM Baclofen + saclofen Calcium-free
Muscimol-stimulate
d wCI
uptaket
58 ± 5 43 47
± 3 ± 4
(26) (19)
51 ± 5’ (12)
buffer
Control
Baclofen,60 M Baclofen, 200 M Baclofen,600 tM
35 37 34 35
± 1 ± 2 (-6) ± 2 (3) ± 2 (0)
Cerebellar microsacs were exposed to the drugs in vitro. Control refers to the action of 3 M muscimol. tNet uptake of 36Cr produced by 30 s exposure to 3 sM muscimol. Values represent nmol Cr mg protein’ 3O s’ and are mean ± SEM, n - 3-4. Values in parentheses represent percent inhibition of muscimol-stimulated uptake produced by baclofen. ‘Significantly different from baclofen alone, P < 0.03 (Scheffe posthoc test after ANOVA).
2469
TABLE 2. Inhibition of muscimol-stimulated chloride uptake by bo.clofen zs not antagonized by a potassium channel blockers TEA and (Ba2’) Treatments
Muscimol-stim
200
TEA,lmM Baclofen
+
36C1
uptaket
26 ± 3 19 ± 2 (27) 19±2 14 ± 2 (27)
Control
Baclofen,
ulated
sM
TEA
± 2 17 ± 1 (39) 50 ± 6 34 ± 4 (32) 28
Control
Baclofen, 200 tMB Ba2’,0.5 mM Baclofen + Ba2’
“Cerebellar microsacs were exposed to the drugs in vitro as described in Methods. Control refers to the action of 3 CM muscimol. bNt uptake of 36C1- produced by 30 s exposure to 3 tM muscimol. Values represent nmol Clmg protein L30 s and are mean ± SEM, n 3. Values in parentheses represent percent inhibition of uptake produced by baclofen.
produced sensitivity
by shorter to
exposure.
muscimol
was
For much
example, greater
the for
apparent
a longer
ex-
posure: a detectable action was observed with 100 nM muscimol, and only 500 nM was required for a half-maximal response at 30 s. This contrasts with an EC50 of about 35 &M at short flux times (10) and raises the possibility that the medium affinity (e.g., 100-500 nM) GABA receptor detected by radioligand binding might be present on a nondesensitizing channel. However, more detailed kinetic studies are required to accurately define the affinity of the nondesensitizing receptor (22). Our preliminary studies of the muscimol-stimulated uptake assayed at 30 s (with or without predesensitization) demonstrate that it is inhibited by bicuculline and picrotoxinin, and is enhanced not only by pentobarbital and flunitrazepam but also by f3-carboline or benzodiazepine inverse agonists (E. Lewin and R. A. Harris, unpublished results). Thus, the nondesensitizing channel may be a variant of the traditional GABA/benzodiazepine receptor complex. These results provide some information about the mechanism by which baclofen inhibits GABAA activity. It is likely due to activation of a GABAB receptor based on the stereospecificity of the action of baclofen, antagonism by 2-OH-saclofen, and dependence on calcium. In addition, the response to baclofen was mimicked by GTP-’y-S, but not by GDP-f3-S, in lysed/resealed membrane vesicles, suggesting mediation by a G protein. This is analogous to the finding of Dolphin et al. (23) that GTP-y-S inhibits neuronal calcium currents in a manner analogous to that of baclofen. However, these investigators also found that GTP--y-S enhanced, and GDP-j3-S reduced, the action of baclofen. We did not observe either of these interactions in our system. The maximum effect of (-) baclofen was seen with 100 &M, the same concentration used to increase P1 production (23) and to inhibit calcium channels (24). However, somewhat lower concentrations are effectivein producing presynaptic inhibition of hippocampal neurons (e.g., maximal effect at 30 fLM) (25). The action of baclofen was blocked by U73122 at a concentration that appears to provide selective inhibition of phospholipase C (15). Subunits of the GABAA receptor can be phosphorylated by protein kinase C (PKC) (26, 27) and this may reduce channel function (28). Our preliminary results indicate that the action of baclofen (i.e., selective inhibition of the slow phase of uptake) is mimicked by phorbol esters that activate PKC, but not by an inactive analog (R. A. Harris, unpublished results).Furthermore, under some
2470
Vol. 5
July 1991
conditions baclofen can increase the turnover of phosphoinositides (23, 29, 30). Taken together, these results suggest that baclofen acts through a GABAB receptor and G protein to increase P1 turnover and thereby activate PKC, leading to inhibition of function by phosphorylation of a subunit of the receptor. However, there are several potential problems with this mechanism. First, although baclofen has been shown to increase P1 turnover (see above), thiseffectis not consistent; for example, baclofen inhibits P1 turnover stimulated by histamine or serotonin without altering basal activity (31, 32). Second, we observed an effect of baclofen at an assay temperature of 0#{176}C, although its action was reduced compared with 34#{176}C,raising the question of whether enzymatic steps are involved. However, some protein kinases are quite active at a temperature of 0#{176}C (33). Further studies are necessary to determine if the phosphorylation mechanism proposed here is valid. The lack of effect of baclofen on GABAA receptor function at short (3-10 s) flux times without preexposure to baclofen is consistent with those of Mehta and Ticku (34) using mouse spinal cord neurons and our previous results with mouse cortex (35). Electrophysiological studies also found no effectsof baclofen on GABAA-mediated currents produced by 0.1- or 0.6-s applications of GABA (36, 37). However, Kardos (8) found that baclofen almost completely inhibited the uptake of 36C1 by cerebellar granule cells at flux times (and baclofen exposure times) of 7 or 14 s. The GABA receptors of cultured fetal granule cells may be different from those of the adult cerebellar membrane vesicles derived from a mixture of cells used in our experiments. For example, Kardos (8) found that muscimol-stimulated uptake of 36Cl reached a maximum at 7 s and declined markedly from 7 to 28 s,whereas we found no decline of uptake over thisperiod. In addition, preincubation with 3 eM GABA produced complete desensitization of the muscimol-stimulated uptake by granule cells (8). These results suggest that cultured granule cells possess an effective system for chloride efflux that may be activated by baclofen and lack nondesensitizing GABAA receptors. Although baclofen inhibited uptake of 36C1 by these cells in a pertussis toxin-sensitive manner, the mechanism appears to be different from that observed in the present LU -C I-
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35
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0 CONTROL
E
30
#{149} BAcLOFEN(200iM)
0
=
0
.01
.1 [U73122],
1
10
pM
Figure 5. Effects of the phospholipase C inhibitor, U73122, on muscimol-stimulated uptake of 6C in the presence and absence of baclofen. The concentration of muscimol was 3 pM, (±)baclofen was 200 pM, and the uptake time was 30 s. Values are mean ± SEM,
n=3-5.
The FASEB Journal
HAHNER
ET AL.
study of cerebellar membrane vesicles where baclofen did not appear to enhance 36C1 efflux, but inhibits a nondesensitizing GABAA channel. The physiological implications of a GABAB receptor inhibiting a nondesensitizing GABAA receptor deserve comment. Receptor desensitization appears to be an important mechanism for terminating synaptic actions of GABA (38), and the GABAB receptor may provide a mechanism for terminating the nondesensitizing receptor. Activation of the nondesensitizing receptor by concentrations of muscimol as low as 100 nM suggests that it could be activated by residual synaptic GABA and provide a tonic inhibition that would be reduced by release of GABA sufficient to activate GABAB receptors. The interaction between GABAA and GABAB receptors could occur only on cells that contain both receptor subtypes, but it is possible that other receptors that activate PKC could produce a similar inhibition of GABAA action, thereby extending the generality of the present results. In summary, these experiments demonstrate that GABA can act both as a neurotransmitter and a neuromodulator on the same membrane vesicle, and emphasize the complexity of GABAergic neurotransmission in brain. We thank Dr. Frank Fitzpatrick for suggesting the use of U73122 in these experiments and for providing this drug, and Drs. Tom Dunwiddie
and
Nancy
Leidenheimer
for helpful
discussions.
This
research was supported by Alcohol, Drug Abuse, and Mental Health Administration grants AA06399 and AA03527, and by the Veterans Administration.
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GABAA
The’
FASFR
Iournal
HAHNIP
T
Al
