Potential Mechanisms Involved in the Negative Coupling Between Serotonin 5-HT 1A Receptors and...

Journal of Neurochemistry Raven Press, Ltd., New York 0 1991 International Society for Neurochemistry

Potential Mechanisms Involved in the Negative Coupling Between Serotonin 5-HTIA Receptors and Carbachol-

Stimulated Phosphoinositide Turnover in the Rat Hippocampus

Y ves Claustre, JesGs Benavides, and Bernard Scatton

Biochemical Pharmacology Group, Synthblabo Recherche (L. E. R.S.), Bagneux, France

Abstract: Serotonin 5-HTIA receptors have been reported to be negatively coupled to muscarinic receptor-stimulated phosphoinositide turnover in the rat hippocampus. In the present study, we have investigated further the phamaco- logical specificity of this negative control and attempted to elucidate the mechanism whereby 5-HTIA receptor activation inhibits the carbachol-stimulated phosphoi nositide response in immature or adult rat hippocampal slices. various 5-HTIA receptor agonists were found to inhibit carbachol (10 p M ) - stimulated formation of total inositol phosphates in immature rat hippocampal slices with the following rank order of potency (IC5,, values in nM): 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (1 1) > ipsapirone (20) > gepirone ( 1 20) > RU 24969 (140) > buspirone (560) > I-(m-trifluo- romethylpheny1)piperazine ( 1,500) > methysergide (5,644); selective 5-HTle, 5-HT2, and 5-HT3 receptor agonists were inactive. The potency of the ~ - H T I A receptor agonists investigated as inhibitors of the carbachol response was well cor- related (r = 0.92) with their potency as inhibitors of the forskolin-stimulated adenylate cyclase in guinea pig hippocampal membranes. 8-OH-DPAT (10 p M ) fully inhibited the carbachol-stimulated formation of inositol di-, tris-, and tetrakisphosphate but only partially antagonized (-40%) inositol monophosphate production. The effect of 8-OH-DPAT on carbachol-stimulated phosphoinositide turnover was not prevented by addition of tetrodotoxin (1 p M ) , by prior de- struction of serotonergic afferents, by experimental manip ulations causing an increase in cyclic AMP levels (addition of 10 p M forskolin), or by changes in membrane potential (increase in K+ concentration or addition of tetraethylam-

monium). Prior intrahippocampal injection of pertussis toxin also failed to alter the ability of 8-OH-DPAT to inhibit the carbachol response. Carbachol-stimulated phosphoinositide turnover in immature rat hippocampal slices was inhibited by the protein kinase C activators phorbol IZmyristate 13- acetate ( 10 p M ) and arachidonic acid ( 100 pM). Moreover, the inhibitory effect of 8-OH-DPAT on the carbachol response was blocked by 10 pM quinacrine (a phospholipase A2 inhibitor) but not by BW 755C (100 pM), a cyclooxygenase and lipoxygenase inhibitor. These results collectively suggest that ~ - H T ~ A receptor activation inhibits carbachol-stimulated phosphoinositide turnover by stimulating a phospholipase Az coupled to 5-HTIA receptors, leading to arachidonic acid release. Arachidonic acid could in turn activate a y-protein kinase C with as a consequence an inhibition of carbachol- stimulated phosphoinositide turnover. This inhibition may be the consequence of a phospholipase C phosphorylation and/or a direct effect on the muscarhic receptor. The latter possibility is supported by the fact that incubation of immature rat hippocampal slices with 8-OH-DPAT (10 p M ) caused a down-regulation (- 16%) of [3H]quinuclidinyl benzylate binding. Key Words: Serotonin 5-HTIA receptors- Phosphoinositide turnover-Hippocampus-Developing rat-Carbachol-8-Hydroxy-2-(di-n-propylamino)tetralin- Protein kinase C-Phospholipase A2-[3H]Quinuclidinyl benzylate binding. Claustre Y. et al. Potential mechanisms involved in the negative coupling between serotonin 5-HTIA receptors and carbachol-stimulated phosphoinositide turnover in the rat hippocampus. J. Neurochem. 56, 1276-1285 (1991).

Physiological responses to serotonin (5-HT) are mediated by several receptors that in binding studies have been classified into three main categories: 5-HT1, 5- HT2, and 5-HT3. The 5-HT1 category has been sub-

Received February 27, 1990; revised manuscript received August 26, 1990; accepted October 9, 1990.

Address correspondence and reprint requests to Dr. B. Scatton at Biochemical Pharmacology Group, SynthClabo Recherche (L.E.R.S.), 31 avenue Paul Vaillant Couturier, 92220 Bagneux, France.

Abbreviafions used: GABA, y-aminobutyric acid; Gi protein, pertussis toxin sensitive inhibitory guanine nucleotide-binding protein;

divided further into four different receptor subtypes, termed 5-HTIA, 5-HTIB, 5-HTlc, and 5-HTfD (Bradley et al., 1986; Peroutka, 1988).

The activation of these receptors is transduced in

H7, I-(5-isoquinolinylsuIfonyl)-2-methylpiperazine; 5-HT, serotonin; IP,, IP,, IP3, and IP4, inositol mono-, bis-, tris-, and tetrakisphosphate, respectively; 8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)tetralin; PBMA, phorbol 12-myristate 13-acetate; PI, phosphoinositide; QNB, quinuclidinyl benzylate; TEA, tetraethylammonium; TFMPP, I-(m- trifluoromethy1phenyl)piperazine; TTX, tetrodotoxin.

1276

SEROTONIN 5-HTlA RECEPTORS AND PI TURNOVER 1277

target cells by several effector systems, such as changes in plasma membrane permeability to potassium, inhibition or activation of adenylate cyclase activity, and inhibition or activation of phosphoinositide (PI) turnover (Peroutka, 1988). The stimulation of the 5-HTIA receptor subtype has been reported to be associated with an increase in the permeability of potassium channels in hippocampal neurons (Andrade and Nicoll, 1987; Colino and Halliwell, 1987) and an inhibition of the forskolin-activated adenylate cyclase in hippocampal membrane preparations as well as in cultured neurons (De Vivo and Maayani, 1986; Bockaert et al., 1987; Dumuis et al., 1988~) . Moreover, we have re- cently provided evidence that 5-HTIA receptors are also negatively coupled to PI phosphodiesterase in the rat hippocampus (Claustre et al., 1 9 8 8 ~ ) . Thus, selective 5-HTIA receptor agonists, e.g., 8-hydroxy-2-(di-n-di- propy1amino)tetralin (8-OH-DPAT) and ipsapirone, inhibit the carbachol-stimulated formation of 3H-inositol phosphates in rat hippocampal (but not striatal or cortical) slices; the effect of 8-OH-DPAT is antagonized by cyanopindolol. Activation of 5-HTlA receptors fails to inhibit the production of 3H-inositol phosphates stimulated by KCI, noradrenaline, or quisqual- ate, thus suggesting that 5-HTIA receptors are negatively coupled to the PI phosphodiesterase specifically activated by muscarinic receptors in the hippocampus (Claustre et al., 1 9 8 8 ~ ) .

The aim of the present study was twofold. First, we wished to investigate further the pharmacological specificity of the PI response to carbachol by studying the effects of various 5-HTlA receptor agonists on the stimulation of accumulation of 3H-inositol phosphates induced by carbachol in immature rat hippocampal slices (the effects of 8-OH-DPAT on the individual inositol mono- and polyphosphates whose release was induced by carbachol were also studied). The second goal of this study was to investigate the mechanism whereby 5-HTlA receptor activation influences carbachol-induced PI turnover stimulation in rat hippocampal slices and in particular to ascertain if the inhibitory effect of 8-OH-DPAT is mediated via a direct interaction between 5-HTIA receptors and phospholipase C or is secondary to changes in another effector mechanism (activation of potassium channels, lowering of cyclic AMP levels, or activation of protein kinases or phospholipase A2). Finally, we explored the possibility that 5-HT1 A receptor stimulation down-regulates muscarinic cholinergic receptors by studying the effect of preincubation with 8-OH-DPAT on the binding of [3H]quinuclidinyl benzylate ([ 3H]QNB) in immature rat hippocampal slices.

MATERIALS AND METHODS

Measurement of PI turnover Immature (8-day-old) and adult (weighing - 150 g)

Sprague-Dawtey (COBS CD strain; Charles River, France) rats were used.

Animals were decapitated, and the brains were dissected out on ice. Hippocampal slices (0.35 mm thick) were prepared by using a McIlwain tissue chopper and preincubated for 1 h in oxygenated (95% 0 2 / 5 % C02) Krebs-Henseleit buffer (composition in mM: NaC1, 118; KCI, 4.7; CaCl,, 2.5; KH2P04, 1.2; MgS04, 1.2, NaHC03, 25; and glucose, 12; pH 7.4) with changes of buffer every 15 min. Labeling of the PI pool with rny~-[~H]inositol (specific activity, 17.1 Ci/ mmol; NEN, Boston, MA, U.S.A.) was performed as described previously (Claustre et al., 19886). In brief, aliquots of slices (- 1 mg of protein) were incubated for 30 min in Krebs solution in the presence of 1 pCi of rny~-[~H]inositol and 5 mM LiCI, after which carbachol was added. In all cases, 5-HTIA receptor agonists were introduced 15 min before carbachol. The incubation was stopped 15 rnin after carbachol addition by adding chloroform/methanoI ( 1 :2 voI/vol); total 3H-inositol phosphates were extracted with water and re- moved from the aqueous phase by ion exchange chromatography with Dowex AG 1 X 8 in the formate form (Brown et al., 1984). After elution, 'H-inositol phosphates were quan- tified by liquid scintillation spectroscopy.

In some experiments, tritium-labeled inositol mono-phosphate (IPJ, inositol bisphosphate (IP2), inositol trisphosphate (IP3), and inositol tetrakisphosphate (IF',) were separated by ion exchange HPLC. In this case, tissue slices were preincubated for 30 rnin in the presence of my~-[~H]inositol (in the absence of LiCl), and the reaction was stopped 2.5 rnin after addition of carbachol and drugs with 300 p1 of 1 M trichloroacetic acid. After standing at room temperature for 20 min and centrifugation, aliquots (500 pl) of supernatants were washed five times with 1 ml of water-saturated diethyl ether and adjusted to pH 7.8 with saturated NaHC03. The 3H-inositol phosphates were separated by a modification of the HPLC method of Irvine et al. (1985) using a SFCC Spherisorb SAX column (0.46 X 35 cm; particle size, 5 pm) (Claustre et al., 19886). The 3H-inositol phosphates were eluted with the following gradient program (1.2 ml/min): a plateau for 5 rnin with 100% water and then a linear gradient of 0-70% 1 Mammonium formate (adjusted to pH 3.7 with orthophosphoric acid) from 5 to 24 min. The buffer concentration was then increased over 1 rnin to a final concentration of 100% 1 M ammonium formate/phosphoric acid (pH 3.7) before replacement with water. Fractions of 600 pl were col- lected, 5 ml of Instagel (Packard, Downers Grove, IL, U.S.A.) was added, and radioactivity was measured by liquid scintillation spectrometry. The identity of the different inositol phosphates was established by comparison with labeled stan- dards.

Binding studies The binding of ~-[benzili~-4,4'-~H]QNB ( [3H]QNB) to

hippocampal slices was performed as described by Jia et al. (1989) and Liles et al. (1986). In brief, hippocampal slices were processed exactly as for the measurement of PI turnover and incubated in a Krebs-Henseleit buffer containing 5 mM LiCl and 100 nM phorbol 12-myristate 13-acetate (PBMA) or 10 pM8-OH-DPAT for 30 min. The reaction was stopped by addition of 3 ml of cold Krebs-Henseleit buffer without LiCl. The slices were then incubated for 2 h at 4°C in 500 pI of a Krebs-Henseleit solution containing 5 nM ['HIQNB (32.9 Ci/mmol; NEN). Nonspecific binding was defined by 100 pM atropine. The binding reaction was stopped by adding 3 ml of ice-cold incubation buffer, followed by aspiration of the medium and by two 5-min washes with 0.5 ml of cold buffer. The slices were then dissolved into 200 b1 of 1 M

J. Neurochem.. Vol. 56, No. 4, 1991

1278 Y. CLAUSTRE ET AL.

NaOH; an aliquot (10 pl) was taken for protein content measurement by the method of Lowry et al. (195 l) , and the re- mainder was used for the measurement of radioactivity by liquid scintillation spectrometry. Specific binding accounted for 60% of total ['HIQNB binding.

Neurotoxic lesions of serotonergic neurons Adult rats, pretreated with desipramine (25 mg/kg i.p.)

and nomifensine (10 mdkg i.p.), 60 and 30 min, respectively, before the neurotoxin injection, were lightly anesthetized with ether and received an intracerebroventricular injection of 5,7- dihydroxytryptamine (200 pd20 p1 of saline). Sham-lesioned animals received the vehicle instead. Measurement of PI turnover was performed 15 days after lesioning. As the carbachol-induced stimulation of hippocampal PI turnover in adult rats is smaller than that in immature rats, a higher (100 pcM) concentration of carbachol was used. The efficacy of the lesion was assessed by measuring 5-HT levels in the frontal cortex by means of HPLC with electrochemical detection according to the technique of Semerdjian-Rouquier et al. (1981).

Local injections of pertussis toxin Adult rats were anesthetized with chloral hydrate (400 ms/

kg i.p.). Pertussis toxin (0.5 pg/l pl of saline) was injected stereotaxically into the left hippocampus at the following co- ordinates: A, -3.8; L, 2.5; H, -4.3 (atlas of Paxinos and Watson, 1982). Sham-lesioned rats received an equivalent volume of saline. Animals were killed 2 days after pertussis toxin infusion, and the hippocampal region (dorsal hippocampus) corresponding to the injected area was dissected out and processed for the measurement of PI turnover.

Chemicals and drugs The following compounds were purchased from Sigma (St.

Louis, MO, U.S.A.): carbachol, atropine, 5,7-dihydroxytryptamine, pertussis toxin, dibutyryl cyclic AMP, PBMA, arachidonic acid, quinacrine, tetrodotoxin (TTX), 1 -(5-isoquin- olinylsulfonyl>2-methylpiperazine (H7), and tetraethylammonium (TEA). Forskolin was purchased from Calbiochem Behring (La Jolla, CA, U.S.A.), staurosporine was from Fluka Chemie AG (Buchs, Switzerland), and NaF was from Prolabo (France). 8-OH-DPAT, BW 755C, gepirone, buspirone, ipsapirone, 1-(M-trifluoromethylpheny1)piperazine (TFMPP),

2-methyl-5-HT, a-methyl-5-HT, methysergide, and RU 24969 were synthesized by our Chemistry Department (Syn- thklabo, Pans, France). CGS 12066A was a generous gill from Ciba-Geigy .

All the compounds were solubilized in water except arachidonic acid and PBMA, which were dissolved in ethanol and diluted in buffer to the desired concentration.

Statistics The statistical significance of the data was evaluated by

variance analysis followed by the Duncan (homogeneous variance) or the Kruskall-Wallis (heterogeneous variance) test.

ICso values were computed from a log-arc sine transfor- mation of the inhibition curves.

RESULTS

Pharmacological specificity of the PI response As was previously observed, among the 5-HT recep-

tor ligands tested, the most active compound was 8- OH-DPAT, which partially inhibited carbachol ( 10 pM)-stimulated PI turnover in immature rat hippocampal slices with an ICso of 11 nM and a maximal decrease of 5 1% (Table 1). Among the other serotonergic ligands examined only those with high affinity for 5-HTIA receptors were able to inhibit this response, with the following rank order of potency: 8-OH-DPAT > ipsapirone > gepirone > RU 24969 > buspirone > TFMPP > methysergide. Agonists specific for 5- HTIB, 5-HT2, and 5-HT3 receptor subtypes were inactive in this model (Table 1).

Effect of 8-OH-DPAT on carbachol-induced stimulation of production of 3H-inositol mono- and polyphosphates

The effects of a high concentration (10 pM) of 8- OH-DPAT on the different metabolic products of the PI response were examined in immature rat hippocampal slices after a short (2.5-min) carbachol(l0 p M ) stimulation period. Under these conditions, 8-OH-

TABLE I . Inhibition by several 5-HT receptor subtype agonists of the carbachol-stimulated PI turnover in hippocampal slices from developing ruts

Maximal 5-HT receptor subtype Drug G o inhibition (70)

S-HTIA 8-OH-DPAT 1 1 (4-31) 51 (42-60) 5-HTlA Ipsapirone 20 (3-165) 28 (22-34)

5-HTIA Buspirone 560 (349-897) 39 (27-5 1) S-HTIJS-HTIB TFMPP 1,500 (200-1 1,280) 53 (38-68) S-HTldS-HTlB ~-HT,J /S -HT~B/~-HT~=/~-HT~ Methysergide 5,644 ( 1,884- 16.9 10) 38 (23-53)

5-HTlA G e p i r o n e 120 (24-600) 39 (34-44)

RU 24969 140 (3.5-5,545) 23 (16-30)

S-HTIB CGS 12066B > 10,000 5-HT2 a-Methyl-5-HT > 10,000 S-HT, 2-Methyl-5-HT > 10,000

Each drug was assayed at five different concentrations. ICso values were computed by taking as the 100% effect the maximal inhibition by each particular drug. The 95% confidence intervals are given in parentheses. Results are derived from at least two different slice preparations.

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SEROTONIN S-HTjA RECEPTORS AND PI TURNOVER 1279

r

800

600

k 400

200

0

0 control

m carbachol(10 PM)

carbachol ( I0 pM) + 8-OH-DPAT (10 p M )

FIG. 1. Inhibition by 8-OH-DPAT of carbachol-stimulated accumulation of 3H-inositol mono- and polyphosphates in hippocampal slices from developing rats. Slices were incubated for 30 min in the presence of my~-[~H]inositol, and carbachol(10 pM) was added. 8-OH-DPAT (1 0 pM) was introduced 15 min before carbachol. The incubation was terminated 150 s after the introduction of carbachol by adding 300 gl of 1 M trichloroacetic acid. Species of 3H-inositol phosphates were separated by ion exchange HPLC. Data are mean k SEM (bars) values obtained in two experiments performed in quadruplicate. Significantly different values are indicated: *p < 0.05 as compared with the carbachol-stimulated group.

DPAT (10 p M ) totally abolished the stimulation by carbachol of the IP2, IP3, and IP4 production but only partially antagonized (as observed under the standard incubation conditions) IP, production (Fig. 1).

Postsynaptic site of action of 8-OH-DPAT To ascertain the direct (postsynaptic) or indirect

(polysynaptic) nature of the 8-OH-DPAT effect, the inhibitory effect of this compound on the carbachol ( 10 p M ) response was examined in the presence of the sodium channel blocker TTX (1 pM). This neurotoxin caused a slight but significant decrease in the carbachol- induced augmentation of PI turnover in immature rat hippocampal slices but failed to affect the inhibitory effect of 8-OH-DPAT (to pM) on this response: without TTX, -36%; with TTX, -37% (Fig. 2).

For obvious methodological reasons, the effect of serotonergic denervation on the 8-OH-DPAT response was examined in hippocampal slices from adult rats. Although the carbachol-induced stimulation of PI turnover in hippocampal slices from unlesioned adult rats was smaller in magnitude than that in developing rats, this effect was almost equally sensitive to 5-HTIA agonists: IC50 and maximal effects for 8-OH-DPAT in the unlesioned adult rat, 72 nM and -42%, respectively. After 5,7-dihydroxytryptamine-induced lesion of serotonergic neurons, the magnitude of the carbachol ( 100 pM)-induced stimulation of PI turnover was greatly increased (434% vs. 157% in control rats), but the inhibitory potency of 8-OH-DPAT on the carbachol

response was not significantly different from that measured in control adult animals: ICSo and maximal effect, 67 nM and -51%, respectively (Table 2). The effec- tiveness of the 5,7-dihydroxytryptamine lesion was at- tested to by a 82% reduction in cortical levels of 5-HT: control, 369 _+ 12 ng/g; lesioned, 66 f 1 1 ng/g ( p

Lack of effect of agents that increase cyclic AMP levels on the 8-OH-DPAT response

To investigate if the inhibition by 5-HTIA receptor stimulation of the carbachol-stimulated PI turnover in immature rat hippocampal slices is secondary to a decrease in cyclic AMP levels, the effect of 8-OH-DPAT ( 1 pM) was measured in the presence of forskolin at a concentration (10 p M ) that increased by 250% the levels of cyclic AMP (data not shown). This forskolin concentration failed to alter the carbachol (10 pM)- evoked stimulation of hippocampal PI turnover and to prevent the inhibition of this response by 8-OH- DPAT (Table 3).

Pertussin toxin prevents the negative coupling of receptors with adenylate cyclase through a blockade of inhibitory guanine nucleotide-binding proteins (Gi proteins). As shown in Fig. 3, in hippocampal slices from adult rats that had received an intrahippocampal injection of pertussis toxin 2 days before the experiment, the carbachol-induced stimulation of PI turnover in the injected region was reduced by -5076, a finding

< 0.01).

15000 -

10000 -

E (1 U

5000 -

+ TTX (1 p M )

T

without TTX

T

0 0 control

carbachol (10 p ~ )

carbachol (10 FM) + 8-OH-DPAT (1 pM)

FIG. 2. Lack of influence of TTX on the effect of 8-OH-DPAT on carbachol-stimulated PI turnover in hippocampal slices from immature rats. Slices were incubated for 30 min in the presence of 1 pCi of rny~-[~H]inositol and 5 mM LiCl and in the presence or in the absence of TTX (1 @). Carbachol(10 f l ) was then added, and the incubation was continued for a further 15 min. 8-OH-DPAT (1 was added 15 rnin before carbachol. Data are mean f SEM (bars) values obtained in three experiments per group, each performed in quadruplicate. Significantly different values are indicated: ' p < 0.01 versus carbachol alone; t p < 0.01 versus the group without TTX.

J. Neuruchem., Vol. 56, Nu. 4, 1991


TABLE 2. Effect of serotonergic denervation on inhibition by 8-OH-DPAT of the carbachol-stimulated PI turnover in hippocampal slices from adult rats

Mean t SEM radioactivity (dpm)

Lesioned Normal

Control 326 f 17 598 f 59 Carbachoi (100 p M ) 837 t 84 3,195 * 241

+ 8-OH-DPAT (0.0 I p M ) 2,829 rfr 122 (-14%) + 8-OH-DPAT (0.1 pM) 702 f 36 (-26%) 2,412 f 95 (-30%)" + 8-OH-DPAT (1 p M ) 2,295 f 198 (-35%,)0 + 8-OH-DPAT (10 u M ~ 1,867 f 231 (-51%)"

803 t 53 (-7%)

687 f 40 (-29) 623 t 51 (-42%)"

Hippocampal slices from control and 5,7-dihydroxytryptamine-lesioned rats were incubated for 30 min in the presence of myo-['H]inositol. Carbachol was added, and the incubation was continued for a further I5 min. 8-OH-DPAT was added 15 min before carbachol. Data were obtained in two experiments performed in quintuplicate. The percent decrease from carbachol alone is given in parentheses.

p < 0.0 1 versus carbachol alone.

attesting to the attenuation of Gi-linked phospholipase C activity. However, this toxin failed to alter the ability of 8-OH-DPAT (10 p M ) to inhibit the carbachol ( 100 pM) response: sham, -40%; with pertussis toxin, -38% (Fig. 3).

Lack of effect of changes in membrane potential on the 8-OH-DPAT response

Depolarization of hippocampal slices from immature rats with 15 mM KCl increased by 1 10% basal PI turnover and potentiated the stimulatory effects of carbachol(l0 p M ) (Fig. 4). However, this increase in K+ concentration failed to affect the inhibitory effect of 8- OH-DPAT (1 p M ) on the carbachol response: control, -41%; with 15 mM KCl, -40% (Fig. 4A). Similar results were obtained with a higher concentration (30 mM) of KCl (data not shown).

Likewise, the K+ channel blocker TEA (0.3 mM) also failed to prevent the 8-OH-DPAT effect, although in the presence of TEA, the carbachol (10 p M ) stimulation by itself was reduced (Fig. 4B).

TABLE 3. Effect offorskolin on inhibition by 8-OH-DPAT of the carbachol-stimulated PI turnover in hippocampal

slices from developing rats

% of control % inhibition

8-OH-DPAT of carbachol Drug Control ( I p M ) response

B-1 1 0 0 + 6 9 8 f 4 Carbachol(10 p M ) 465 f 50 331 f 28" -37

+ Forskolin (10 uM) 5 10 t 5 1 366 t 25' -35

Slices were preincubated for 30 min in the presence of my+ ['H]inositol and forskolin (10 p M ) and for I5 min in the presence of 8-OH-DPAT (1 p M ) . The reaction was stopped 15 min after carbachol addition (10 pM). Data are mean -C SEM values obtained in three experiments performed in quadruplicate. Basat values are 1, I28 f 70 dpm.

p < 0.01 versus the control group.

Possible involvement of protein kinase C and phospholipase A2 in the 8-OH-DPAT response

Addition of 10 pM PBMA, a protein kinase C activator, caused a reduction in the carbachol (10 pM) stimulation of PI turnover in immature rat hippocampal slices that was similar in magnitude to that seen with 8-OH-DPAT ( 1 p M ) (Fig. 5A).

Arachidonic acid (100 p M ) also inhibited the carbachol-induced stimulation of PI turnover in immature rat hippocampal slices to a similar extent as %OH-

SHAM PERTUSSIS TOXIN

I T

-- 0 control

carbachol(100pM) + E-OH-DPAT(10 pM) carbachol(100 pM)

FIG. 3. Pertussis toxin fails to affect the inhibition by 8-OH-DPAT of carbachol-stimulated PI turnover in hippocampal slices from adult rats. The effect of 8-OH-DPAT (1 0 p.A4) on the carbachol(lO0 pnn)- stimulated PI turnover was measured in hippocampal slices (prepared from the injected region) from adult rats 2 days after an intrahippocampal injection of pertussis toxin (0.5 pg) or saline as described in Materials and Methods. Data are mean * SEM (bars) values obtained in two experiments performed in sextuplicate. Significantly different values are indicated: ' p < 0.01 versus carbachol alone; t p -= 0.01 versus sham-lesioned rats. Basal values were 1,035 f 61 dprn.

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SEROTONIN ~-HT,.,, RECEPTORS AND pr TURNOVER 1281

A 0 effect of 8-OH-DPAT on the carbachol-stimulated PI turnover (Fig. 6).

Staurosporine (10 nM) was unable to prevent the inhibitory effect of 8-OH-DPAT, PBMA, or arachidonic acid on the carbachol response (Table 4). This 8-OH-DPAT effect was also resistant to H7: control, 1,195 k 102 dpm; carbachol (10 pM), 4,696 k 484 dpm; carbachol plus 8-OH-DPAT (1 pM), 3,520 -t 250 dpm; and carbachol plus 8-OH-DPAT plus H7 (100

0 Conlrol Carbachol (lo p ~ )

Carbachol (10 pM) + SOH-DPAT (1 PM)

FIG. 4. Lack of effect of (A) KCI (15 mM) and (9) TEA on the inhibition by 8-OH-DPAT of carbachol-stimulated PI turnover in hippocampal slices from developing rats. Slices were incubated for 30 min in the presence of my~-[~H]inositol. after which carbachol (1 0 &l) was added. 8-OH-DPAT and KCI or TEA were introduced 15 and 30 min, respectively, before carbachol. The increase in KCI concentration was ismsmotically compensated for by a concom- itant decrease in NaCl concentration. Data are mean k SEM (bars) values obtained in two experiments performed in quintuplicate. Significantly different values are indicated: *p < 0.01 versus the respective carbachol groups; tp < 0.01 versus 5 mM K+ controls or controls without TEA.

DPAT (Fig. 5B). Moreover, the inhibitory effect of 8- OH-DPAT (1 pM) on the carbachol response was almost totally prevented by quinacrine (10 a), an inhibitor of phospholipase A2 (Fig. 5C). In contrast, BW 755C (100 pM), an inhibitor of both cyclooxygenase and lipoxygenase activities, was unable to prevent the

A B

900 r T 600 r T

pM), 3,287 rfr 334 dpm.

Effect of 8-OH-DPAT on [3H]QNB binding to immature rat hippocampal slices

Incubation of hippocampal slices from immature rats for 30 min in the presence of 8-OH-DPAT (10 p M ) caused a significant reduction (- 16%) of [3H]QNB binding. Similarly, incubation of hippocampal slices in the presence of 0.1 pM PBMA reduced by 13% [3HJQNB binding (Table 5).

DISCUSSION

The existence of a negative coupling between 5-HTIA receptors and carbachol-stimulated PI turnover was suggested by previous studies from our laboratory (Claustre et al., 19880) in which it was shown that two 5-HTIA agonists (8-OH-DPAT and ipsapirone) but not a 5-HT2 (a-methyl-5-HT) or a 5-HT3 (Zmethyl-5-HT) agonist partially antagonized the PI turnover stimulation caused by carbachol in adult and immature rat hippocampal slices. This view is further substantiated

C

r * i

- n Control

Carbachol (10 pM)

Carbachol (10 pM)+B-OH-DPAT(l PM)

+ Quinacrine (10 pM)

Carbachol (10 pM)

Carbachol (10 $4) + PBMA(10pM) + Arachidonate (100 pM) Carbachol (10 pM)+B-OH-DPAT(lpM)

I /J control w Carbachol (10 pM)

Carbachol (10 pM)

FIG. 5. Inhibition by (A) PBMA and (9) arachidonic acid of carbachol-stimulated PI turnover in hippocampal slices from immature rats and (C) reversal of the inhibitory effect of 8-OH-DPAT by quinacrine. Slices were incubated for 30 min in the presence of my~-[~H]inositol, after which carbachol(l0 pM) was added. 8-OH-DPAT, arachidonic acid, and PBMA were introduced 15 min before carbachol. Quinacrine was added 30 min before carbachol. Data are mean k SEM (bars) values obtained in at least two experiments performed in quintuplicate. Significantly different values are indicated: *p < 0.05 as cornpared with the carbachol group (A and B) or as compared with the carbachol plus 8-OH-DPAT group (C). Basal values were 1,024 -+ 1 15 dpm.

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Y. CLAUSTRE ET AL.

TABLE 5. Effect o f 8-OH-DPAT and PBMA on 13H]QNB binding to immature rat hippocampal slices

1

*

Control CARE CARB CARB i I O r M ) + +

(1 r M ) + 8-OH-DPAT 8-OH-DPAT

0W755C (100 uM1

FIG. 6. Lack of antagonism by BW 755C of the inhibitory effect of &OH-DPAT on carbachol-stimulated PI turnover in hippocampal slices from developing rats. Slices were incubated for 30 min in the presence of rny~-[~H]inositd, after which carbachd was added. 8-OH-DPAT and BW 755C were added 15 and 30 min before carbachol, respectively. Data are mean f SEM (bars) values obtained in one experiment performed in quintuplicate. Significantly different values are indicated: *p < 0.01 versus carbachol alone. Basal values were 755 f 96 dpm.

by the results of the present study, which show that other specific or nonspecific 5-HTIA agonists (ipsapirone, gepirone, buspirone, RU 24969, TFMPP, and methysergide) diminish, with varying potencies, the carbachol response. The potency of the ~ - H T I A receptor agonists investigated for the inhibition of the carbachol-stimulated PI turnover correlates well ( r = 0.92) with the potencies of these drugs at inhibiting the forskolin-stimulated adenylate cyclase in guinea pig membranes (Bockaert et al., 1987), a finding lending

['HIQNB binding (fmol/mg of protein)

Control 5 1 2 t 30 8-OH-DPAT (10 p M ) 428 t 24 (-16%)"

Ethanol (0.01%) PBMA (0.1 p M )

590 k 24 511 t20( -13%)"

Slices were incubated for 2 h at 4°C in the presence of 5 nM ['HIQNB. 8-OH-DPAT and PBMA were added 30 min before ['HIQNB. PBMA was dissolved in 0.01% ethanol. Data are mean f SEM values of three independent experiments performed in quadruplicate.

' p < 0.05 versus the respective control.

support to the view that the ~ - H T ~ A receptor subtype is negatively coupled to the carbachol-stimulated PI turnover in the rat hippocampus.

The negative modulation of the carbachol response by 5-HTIA receptor agonists seems to be brought about at the postsynaptic level, as the 8-OH-DPAT effect was still observed in the presence of TTX or in slices from rats deprived of their serotonergic innervation by prior administration of 5,7-dihydroxytryptamine. These results strongly suggest that muscarink and ~ - H T I A receptors are colocalized on the same neurons in the hippocampus. A possible anatomical substrate for this interaction could be the hippocampal pyramidal cells. Indeed, autoradiographic studies have revealed high densities of both muscarink (Spencer et al., 1986) and ~ - H T ~ A (Pazos and Palacios, 1985; VergC et al., 1986) receptors on these cells. Moreover, electrophysiological studies have demonstrated that 5-HTIA receptor agonists are able to block the acetylcholine-evoked increase in the firing rate of hippocampal pyramidal neurons (Martin and Mason, 1987).

The inhibitory effect of 8-OH-DPAT on the carbachol-induced stimulation of formation of total inositol phosphates is never greater than 40-50%; this

TABLE 4. Inability of staurosporine to prevent the inhibition by 8-OH-DPA T, PBMA, or arachidonic acid of carbachol-stimtilaied PI turnover in

hippocampal slices from immature rats

Mean f SEM radioactivity (dpm)

Staurosporine Control (10 nM)

Basal 1,174 t 49 1.179 f 94 Carbachol ( 10 p M ) 5,744 * 269 6,151 C 794

+ 8-OH-DPAT ( 1 p M ) + PBMA (100 p M )

4,290 t 215 (-32%)" 4,339 f 279 (-31%)"

4,106 f 240 (-42%)" 4,467 f 344 (-32%)'

+ Arachidonic acid (100 p M ) 4,499 C 277 (-27%)" 4,382 t 327 (-35%)"

Staurosporine (10 nM) was added 15 min before 8-OH-DPAT ( I pM), PBMA (100 pM), or arachidonate (100 p M ) and 30 min before carbachol (10 p M ) . Data were obtained in two to four experiments performed in quintuplicate. The percent decrease from carbachol alone is given in parentheses.

p < 0.01, 'p < 0.05 versus carbachol alone.

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SEROTONIN S-HTIA RECEPTORS AND PI TURNOVER 1283

could suggest that not all muscarinic receptors are under the negative influence of 5 - H T 1 ~ receptors. How- ever, the fact that 8-OH-DPAT inhibits totally [3H]IP2, [3H]IP3, and [3H]IP4 production but only partially [3H]IP, accumulation caused by carbachol argues against such an interpretation. The partial inhibition by 8-OH-DPAT of the carbachol-stimulated formation of the total pool of inositol phosphates may be due to the fact that inositol polyphosphate production and the IP1 formed from inositol polyphosphates are con- trolled negatively by ~ - H T I A receptors, whereas the direct formation of IPI from phosphatidylinositol is not sensitive to this modulation. This hypothesis would be compatible with previous studies showing that inositol mono- and polyphosphates are synthesized via different pathways that are subject to different regulatory influences (Majerus et al., 1986). As the inositol polyphosphates (mainly IP3 and IP4) are the second messenger species, it appears from our data that the carbachol- induced formation of all those inositol phosphates able to affect cellular calcium homeostasis (Downes, 1988; Berridge and Irvine, 1989) is under a 5-HTIA receptor negative control.

One of the main purposes of the present study was to investigate the mechanism by which ~-HTIA receptor activation inhibits muscarinic receptor-induced stimulation of PI turnover in the rat hippocampus. The reduction in [3H]QNB binding observed in hippocampal slices incubated in the presence of 8-OH-DPAT suggests that a down-regulation of muscarinic receptors may, at least partially, be responsible for the PI turnover inhibition induced by 5-HTIA receptor stimulation. Such a mechanism has been advanced to explain the inhibitory effects of phorbol esters on muscarinic responses (Jia et al., 1989). However, the limited decrease of [3H]QNB binding induced by 8-OH-DPAT suggests that down-regulation of muscarinic receptors is not the only operative mechanism involved in the S-HT~A receptor-mediated inhibition of muscarinic responses.

The activation of ~ - H T I A receptors is transduced by at least three effector mechanisms: PI turnover inhibition (Claustre et al., 1988a), adenylate cyclase inhibition (De Vivo and Maayani, 1986; Bockaert et al., 1987; Dumuis et al., 1988a), and increase in potassium channel conductance leading to a neuronal hyperpolarization (Andrade and Nicoll, 1987; C o h o and Hal- liwell, 1987). The two latter effector mechanisms ap- pear to require a coupling guanine nucleotide binding protein (Harrington et al., 1988; Innis et al., 1988). In rats injected with pertussis toxin, the carbachol-induced stimulation of hippocampal PI turnover was blunted, as expected from electrophysiological and biochemical studies demonstrating a coupling of muscarinic receptors to a pertussis toxin-sensitive guanine nucleotide binding protein (Cockcroft, 1987). However, this toxin failed to alter the inhibitory effect of 8-OH-DPAT on carbachol-stimulated PI turnover, thus indicating that if a coupling protein is necessary for the PI turnover inhibition, this protein is less sensitive (or insensitive)

to pertussis toxin than those mediating the PI turnover activation.

In the cell, the different second messengers are prob- ably under mutual regulatory influences (Enna and Karbon, 1987). Thus, the inhibition of carbachol- stimulated inositol phosphate production caused by 5- HTIA receptor stimulation may be secondary to changes in other transduction mechanisms, such as the 5-HTIA receptor-mediated adenylate cyclase inhibition. However, an experimental manipulation aimed at increasing cyclic AMP levels (activation of adenylate cyclase by forskolin) failed to reverse the inhibitory effect of 8-OH-DPAT on the carbachol response. Moreover, as mentioned above, pertussis toxin, which blocks the Gi protein required for the negative coupling of receptors to adenylate cyclase, did not prevent the 8-OH- DPAT inhibitory effect. An additional argument against the involvement of cyclic AMP is the obser- vation that this second messenger inhibits rather than enhances al-receptor-mediated PI hydrolysis stimulation (Neylon and Summers, 1988).

The activation of 5-HTIA receptors also results in neuronal hyperpolarization due to opening of potassium channels ( C o h o and Halliwell, 1987). Because hyperpolarization causes the inactivation of voltage- sensitive calcium channels and because PI turnover activation induced by carbachol has been shown to be calcium dependent (Kendall and Nahorski, 1984), the possibility had to be considered that the inhibition of the inositol phosphate production is mediated by changes in the neuronal membrane permeability to cations. Our results do not lend support to this mechanism because the inhibitory effect of 8-OH-DPAT on carbachol-stimulated PI turnover in hippocampal slices was still observed when neurons were depolarized by increasing potassium concentrations or when only mi- cromolar concentrations of calcium were present in the incubation medium (Y. Claustre, unpublished data). Moreover, TEA, a nonspecific potassium channel blocker, was unable to overcome the 8-OH-DPAT inhibitory effect. Thus, the 8-OH-DPAT-induced inhibition of PI turnover does not seem to be mediated by changes in membrane potential or intracellular calcium concentrations. However, a reciprocal mechanism whereby the PI turnover inhibition could result in the changes in ionic permeability observed during ~ - H T I A receptor stimulation remains a possibility.

It has been reported that protein kinase C-catalyzed phosphorylation of phospholipase C results in an inhibition of PI turnover (Labarca et al., 1984). Such a phosphorylation mechanism has been proposed to explain the inhibitory effect of y-aminobutyric acide (GABAB) receptor agonists on the carbachol-stimulated PI turnover in the mouse cerebral cortex (Godfrey et al., 1988). A similar mechanism may account for the inhibitory effect of 5-HTIA receptor stimulation on carbachol-stimulated PI turnover, because this latter inhibitory effect was mimicked by the protein kinase C activator PBMA. A likely effector for this protein

J. Neurochem., Vol. 56. No. 4, 1991


kinase C activation is arachidonic acid. Recent studies (Naor et al., 1988; Nishizuka, 1988) have indeed demonstrated that arachidonic acid specifically activates the y-protein kinase C subspecies, which is highly en- riched in the hippocampus, and arachidonic acid can be generated by the operation of a receptor-activated phospholipase A2 (Axelrod et al., 1988). The facts that in rat hippocampal slices arachidonic acid mimicked the 8-OH-DPAT-induced inhibition of carbachol- stimulated PI turnover and that quinacrine, a phospholipase A2 inhibitor (Duman et al., 1986), prevented the inhibitory effect of 8-OH-DPAT on this response suggest that this phospholipid hydrolysis product may be implicated in the S-HTIA receptor-mediated inhibition of muscarinic receptor-induced formation of inositol phosphates. The proposed inhibitory mechanism would require the existence of a positive coupling between 5-HTlA receptors and phospholipase Az, as has been observed for other receptors negatively coupled to PI turnover, such as the N-methyl-Baspartate and GABAB receptors (Duman et al., 1986; Dumuis et al., 19886).

Thus, the existing experimental evidence is compatible with a mechanism by which the activation of 5-HTIA receptors would result in arachidonic acid release via the operation of a phospholipase A2 positively coupled to 5-HTlA receptors. Released arachidonic acid would in turn activate a protein kinase C, with this activation resulting in partial blockade of the carbachol- stimulated PI turnover, mediated by either phospholipase C inhibition or muscarinic receptor down-regulation (Fig. 7). Alternatively, as proposed for adre- nocorticotropin by Schrama et al. (1986), the protein kinase C-catalyzed phosphorylation of a B50 protein, which leads to a decrease in the phosphoinositol phosphate kinase activity, may provide a molecular mechanism for the ~ - H T ~ A receptor-mediated inhibition of PI turnover. This mechanism appears compatible with the presently observed preferential inhibition of the inositol polyphosphate formation by 8-OH-DPAT. This regulatory cascade seems to be rapid, as the inhibitory potency of 8-OH-DPAT is not reduced by de-

Mi Receptor

~ - H T > A Receptor

Phospholipids acid

DAG

FIG. 7. Hypothetical mechanism whereby 5-HTIA receptor activation could lead to diminished muscarinic receptor-mediated formation of inositol phosphates. DAG, diacylglycerol; G, guanine nucleotide-binding protein; HETE, hydroxyeicosatetraenoic acid; LT, leukotriene; PG. prostaglandin; PIP2, phosphatidylinositol bisphosphate.

creasing or eliminating the preincubation period with this ~ - H T I A agonist (Y. Claustre, unpublished data).

Support for the direct implication of arachidonic acid rather than one of its metabolites in this regulatory cascade is provided by experiments in which an inhibitor of the cyclooxygenase and lipoxygenase activities (BW 755C) was unable to antagonize the inhibitory effect of 8-OH-DPAT on the carbachol response.

Overall, our results suggest that the most likely mechanism involved in the negative coupling between 5-HTIA receptors and muscarinic receptor-stimulated PI turnover is a phospholipase Az-mediated arachidonic acid release. Arachidonic acid would in turn activate y-protein kinase C, leading to a decrease in the muscarinic responses. The anatomical correlation observed between the autoradiographic distributions of 5-HTIA receptors (as labeled by [3H]8-OH-DPAT) and of protein kinase C (as labeled by [3H]PBMA) (Pazos and Palacios, 1985; Vergk et al., 1986; Worley et al., 1986) would be compatible with this hypothesis. Ob- viously, an unequivocal demonstration of this potential mechanism will require the use of specific y-protein kinase C blockers. In preliminary studies with two inhibitors of protein kinase C (H7 and staurosponne), we have been unable to prevent the inhibitory effect of 8-OH-DPAT on carbachol-stimulated PI turnover by H7 (10 p M ) and of g-OH-DPAT, PBMA, and arachidonic acid by staurosporine (10 nM). However, because the activity of these protein kinase C inhibitors has never been established in hippocampal slices and inasmuch as electrophysiologid studies have provided evidence for effects of PBMA and diacylglycerol being insensitive to H7 and staurosporine (Hockberger et al., 1989), the failure of these compounds to block the 8- OH-DPAT effects does not preclude the operation of the above-proposed mechanisms.

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Potential Mechanisms Involved in the Negative Coupling Between Serotonin 5-HT 1A Receptors and...

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