©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Subtype of the Prostacyclin Receptor Expressed in the Central Nervous System (*)

(Received for publication, June 9, 1995; and in revised form, November 27, 1995)

Hajime Takechi (1) (2) Kiyoshi Matsumura (1) (2) Yumiko Watanabe (1) (2) Koichi Kato (3) Ryoji Noyori (3) Masaaki Suzuki (1) (4) Yasuyoshi Watanabe (1) (2)(§)

From the  (1)Subfemtomole Biorecognition Project, Research Development Corporation of Japan, Osaka 565, the (2)Department of Neuroscience, Osaka Bioscience Institute, Osaka 565, the (3)Department of Chemistry, Faculty of Science, Nagoya University, Nagoya 464-01, and the (4)Department of Applied Chemistry, Faculty of Engineering, Gifu University, Gifu 501-11, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

By use of several prostacyclin analogs and an in vitro autoradiographic technique, we have found a novel subtype of the prostacyclin receptor, one having different binding properties compared with those of the known prostacyclin receptor in the rat brain. Isocarbacyclin, which is a potent agonist for the known prostacyclin receptor, had high affinity for the novel subtype (dissociation constant (K) of 7.8 nM). However, iloprost, which is usually used as a stable prostacyclin analog, showed low affinity binding (K = 159 nM) for the subtype. Other prostaglandins showed no or little affinity for the subtype. [^3H]Isocarbacyclin binding was high in the thalamus, lateral septal nucleus, hippocampus, cerebral cortex, striatum, and dorsal cochlear nucleus. Although the nucleus of the solitary tract and the spinal trigeminal nucleus showed a high density of [^3H]isocarbacyclin binding, [^3H]iloprost also had high affinity in these regions, and the binding specificity was similar to that for the known prostacyclin receptor. Hemilesion studies of striatal neurons lesioned by kainate or of dopaminergic afferents lesioned by 6-hydroxydopamine revealed that the binding sites of the novel subtype exist on neuronal cells in the striatum, but not on the presynaptic terminal of afferents or on glial cells. Electrophysiological studies carried out in the CA1 region of the hippocampus revealed that prostacyclin analogs have a facilitatory effect on the excitatory transmission through the novel prostacyclin receptor. The widespread expression of the prostacyclin receptor in the central nervous system suggests that prostacyclin has important roles in neuronal activity.


INTRODUCTION

Prostaglandins (PGs) (^1)and thromboxane are formed from arachidonic acid by cyclooxygenase and the respective synthase for each PG, such as PGD synthase and PGI(2) synthase. These prostanoids exert a variety of functions through their specific membrane receptors coupled to G proteins not only in the peripheral organs, but also in the central nervous system. For example, PGD(2) acts as a sleep inducer(1, 2) , produces hypothermia (3) , inhibits luteinizing hormone-releasing hormone release(4) , and is involved in biphasic modulation of pain sensation (5) and modification of olfaction(6) . PGE(2) has hyperthermic(7) , sedative(8) , anticonvulsive(9, 10) , and antidiuretic effects (11) and induces wakefulness(12, 13) , stimulates luteinizing hormone-releasing hormone release(14) , modifies pain(5, 9) , and regulates food intake(11) . Also, PGF has an antidiuretic effect (15) and an inhibitory effect on oxytocin release(16) . These three PGs are reported to be involved in the modulation of neurotransmitter release as well(17) . Prostacyclin, discovered in 1976, is known to be a potent vasodilator and also to have an inhibitory effect on platelet aggregation(18) . In the brain, however, it is not clear whether prostacyclin has specific functions or not. It seems that the chemical instability of prostacyclin and lack of knowledge about the prostacyclin receptor in the brain have hampered the investigation.

Recently, we reported the presence and distribution of the prostacyclin receptor in the central nervous system by use of [^3H]iloprost, a stable prostacyclin analog, and an in vitro autoradiographic technique(19) . High density binding was observed in the nucleus of the solitary tract (NTS) and in the spinal trigeminal nucleus in the lower brain stem as well as in the dorsal horn of the spinal cord. Much weaker but significant binding was found in various other regions of the brain such as the thalamus and cerebral cortex.

In addition to iloprost, there are several stable prostacyclin analogs, including isocarbacyclin(20) . When [^3H]isocarbacyclin was preliminarily used in the autoradiographic study instead of [^3H]iloprost, we found that its binding potency was much higher than that of [^3H]iloprost in the thalamus and cerebral cortex, whereas both ligands showed comparable binding in some regions of the medulla and spinal cord. This observation suggested the existence of at least two distinct prostacyclin receptors in the brain, with isocarbacyclin binding to both, but iloprost preferentially binding to just one of the two. In this study, we examined in detail selected properties of the [^3H]isocarbacyclin-binding site in the rat brain with special attention to its distinction from those of the [^3H]iloprost-binding site. We demonstrated thereby that most of the prostacyclin receptors in the brain are distinct from the previously known prostacyclin receptor.


EXPERIMENTAL PROCEDURES

Materials

[^3H]Iloprost, iloprost, and autoradiographic [^3H]micro-scale were purchased from Amersham Corp. [^3H]SCH23390 and [^3H]BTCP were from Dupont NEN. SCH23390 and GBR12909 were purchased from Research Chemicals Inc. [^3H]Isocarbacyclin (21) and isocarbacyclin (TEI7165) were kindly provided by Dr. Seizi Kurozumi (Teijin Ltd., Tokyo). Carbacyclin, PGE(1), PGE(2), PGF, PGD(2), 6-hydroxydopamine, and kainate were purchased from Sigma. Cicaprost was from Prof. Günter Stock (Schering Aktiengesellschaft, Berlin). Forskolin and 3-isobutyl-1-methylxanthine were purchased from Wako (Osaka, Japan).

Animals

Male Wistar rats weighing 190-220 g were used for the receptor binding and lesion studies, and those weighing 150-200 g were used for the measurement of cAMP.

Receptor Binding Study

In vitro quantitative autoradiography was performed on the brains of male Wistar rats as described previously(19) . Briefly, the rats were anesthetized with pentobarbital (50 mg/kg) and perfused via the left ventricle with cold 10 mM sodium phosphate-buffered saline (pH 7.4). Frozen serial sections of 10 µm thickness were cut with a cryostat at -20 °C and mounted on gelatin-coated glass slides. All binding procedures were done at 4 °C, unless otherwise stated. The sections were preincubated in three changes of 50 mM Tris-HCl (pH 7.4) containing 10 mM MgCl(2) for a total of 60 min. Then the sections were incubated with 10 nM [^3H]isocarbacyclin (except for the saturation experiment for the Scatchard plot analysis) in the same buffer. [^3H]Iloprost was also used for the Scatchard plot analysis. After the incubation, the sections were rinsed by four sequential short dips (30 s each) in the same buffer and air-dried. Nonspecific binding was determined using consecutive sections with an excess amount of unlabeled isocarbacyclin (10 µM) in the incubation mixture. After having been dried in a desiccator, the slides were exposed to tritium-sensitive film (Hyperfilm-^3H, Amersham Corp.) or an imaging plate (BAS TR2040, Fuji Film Co., Ltd., Tokyo). After calibration with plastic tritium scales, densitometric analysis of the autoradiographs was performed with NIH image analysis software(22) . From the specific radioactivities of ^3H-labeled ligands, optical densities were converted into femtomoles/milligram of tissue.

Lesion Study

Chemical lesions with either kainate or 6-hydroxydopamine were made under pentobarbital anesthesia (50 mg/kg). Wistar rats were positioned in a stereotaxic apparatus, and the chemical solutions were injected locally according to the atlas of Paxinos and Watson (23) at a rate of 0.2 µl/min via a stainless steel cannula (0.4-mm diameter) implanted into the brain and connected to a microinfusion pump. Striatal neuronal cell bodies were lesioned by unilateral injections of kainate (2 µl each, 0.1%, adjusted to pH 7.5 with 1 M NaOH) both in the anteromedial (+1.0 AP, 2.5 ML right, -5.5 DV) and in the posterolateral (-0.4 AP, 3.5 ML right, -5 DV) parts of the neostriatum. The nigrostriatal dopamine neurons were destroyed unilaterally by injection of 6-hydroxydopamine (8 µg in 2 µl of water containing 0.1% ascorbic acid) into the medial forebrain bundle (-2.8 AP, 2.0 ML right, -8.3 DV). Three weeks after the injection, the animals were sacrificed, and cryostat sections were made as described above. In the case of 6-hydroxydopamine lesioning, the effectiveness of the lesioning was examined by assessment of the contralateral circling response to apomorphine (0.5 mg/kg subcutaneously) 5 days before sacrifice. Only animals displaying contralateral rotations (>45 rotations in 5 min) were used in the autoradiographic study. As references for [^3H]isocarbacyclin binding in the lesioned rats, binding studies with [^3H]SCH23390 (3 nM) for the dopamine D(1) receptor and [^3H]BTCP (1 nM) for the dopamine uptake site were performed with the consecutive sections. Binding buffer solutions contained Tris-HCl (50 mM, pH 7.4), NaCl (100 mM), KCl (5 mM), CaCl(2) (2 mM), and MgCl(2) (1 mM) for [^3H]SCH23390 and sodium phosphate buffer (50 mM, pH 7.4) for [^3H]BTCP. In the case of the binding with [^3H]BTCP, the sections were rinsed with a buffer containing Tris-HCl (10 mM, pH 7.4) and NaCl (50 mM). To determine nonspecific binding, we employed excess amounts of the unlabeled ligands, except in the case of [^3H]BTCP binding, where an excess amount of GBR12909 was used.

cAMP Measurement

Male Wistar rats were killed, and their brains were rapidly removed and dissected on ice. Cross-chopped slices (350 times 350 µm) of cerebral cortex were prepared with a McIlwain tissue chopper and incubated at 37 °C for 30 min in Krebs-Ringer bicarbonate buffer (113 mM NaCl, 3 mM KCl, 2 mM CaCl(2), 1 mM MgCl(2), 1 mM NaH(2)PO(4), 11 mM glucose, and 25 mM NaHCO(3)) previously gassed with 95% O(2), 5% CO(2). Next, the buffer was replaced with Krebs-Ringer bicarbonate buffer containing 50 µM 3-isobutyl-1-methylxanthine and incubated for 30 min. Aliquots of the slice suspension (400 µl) were then distributed into individual test tubes, and drug solutions (8 µl) were added. In the experiments to test the inhibitory effect on adenylate cyclase, forskolin was used at a final concentration of 10 µM. After having been gassed with a stream of 95% O(2), 5% CO(2), the tubes were capped and incubated at 37 °C for 15 min. The reaction was terminated by adding 100 µl of 1 N HCl. The slice suspension was then homogenized and centrifuged (10,000 times g for 10 min). The supernatant was collected, and the cAMP level was determined in duplicate with a cAMP enzyme immunoassay system (Amersham International, United Kingdom). Protein contents were then determined in duplicate in the pellet by the method of Bradford (24) .

Electrophysiological Recording

Transverse hippocampal slices (400 µm) were prepared from 3-4-week-old male Wistar rats and maintained by standard procedures (temperature of 31 ± 0.5 °C, submerged recording, and flow rate of 1.5-2 ml/min). The composition of the external recording solution was 113 mM NaCl, 3 mM KCl, 2 mM CaCl(2), 1 mM MgCl(2), 1 mM NaH(2)PO(4), 11 mM glucose, and 25 mM NaHCO(3), and the solution was gassed with 95% O(2), 5% CO(2). Field excitatory postsynaptic potentials (EPSPs) were elicited by stimulation of the Schaffer collateral-commissural afferents with a bipolar tungsten electrode (once every 10 s) and were recorded with a glass pipette (1-5 megaohms, filled with 3 M NaCl) placed in the stratum radiatum of the CA1 region. Six consecutive sweeps were averaged, and the initial EPSP slope was measured. Stimulus intensity was adjusted to produce a response of 0.1 mV. Mean EPSP slope values for the 10 min preceding the application of prostacyclin analogs were used as base-line values.


RESULTS

In the present binding experiments, fresh-frozen tissues and in vitro quantitative autoradiography were used since small regions, such as the NTS, could be easily investigated with this technique. Fig. 1shows the time course of [^3H]isocarbacyclin binding in the cryostat sections of the rat brain. At 25 °C, the specific binding of [^3H]isocarbacyclin reached equilibrium as early as 5 min, whereas it took >120 min to reach a plateau at 4 °C. Although the specific binding after reaching equilibrium was almost the same at both temperatures, nonspecific binding of [^3H]isocarbacyclin was lower at 4 °C. Therefore, we used the incubation condition of 120 min at 4 °C in the subsequent experiments. We also observed that binding was reversible at 4 °C (Fig. 1).


Figure 1: Time course of [^3H]isocarbacyclin binding. Cryostat sections were incubated with 10 nM [^3H]isocarbacyclin at 25 °C (closed symbols) for 5, 10, 20, 40, or 60 min or at 4 °C (open symbols) for 5, 10, 30, 60, 120, or 180 min. Dissociation was observed in buffer containing 10 µM unlabeled isocarbacyclin after a 60-min incubation with 10 nM [^3H]isocarbacyclin. Total (bullet, circle), specific (, box), and nonspecific (, up triangle) binding and dissociation (times) in the thalamic region were quantitatively analyzed and plotted. Values are means of triplicates in a single experiment. Error bars show S.E. (where visible) for total and nonspecific binding.



In our previous study(19) , [^3H]iloprost binding in the NTS was much higher than that in the thalamus when 20 nM [^3H] iloprost was used, two times higher than the known K(d) value (Fig. 2A). The binding study using 10 nM [^3H]isocarbacyclin showed, however, relatively equal amounts of binding in both the NTS and thalamus (Fig. 2C). Most of the other regions, such as the cerebral cortex and striatum, showed a similar tendency of binding by both agents as the thalamus. Therefore, we compared in detail the binding properties mostly in the NTS and thalamus. To analyze the binding properties of [^3H]isocarbacyclin and [^3H]iloprost, we performed Scatchard plot analysis. Scatchard analysis carried out using [^3H]isocarbacyclin showed single high affinity sites in the NTS, thalamus, and striatum, with K(d) values of 3.9, 7.8, and 8.9 nM, respectively (Fig. 3A). K(d) values for both the thalamus and striatum were thus slightly higher than the value for the NTS. The difference was much more evident when [^3H]iloprost was used as a ligand (Fig. 3B). For the NTS, the K(d) was 6.8 nM, whereas that for the thalamus was very large (159 nM). It is important to note that high affinity binding sites for [^3H]iloprost were not observed in the thalamus (Fig. 3B). B(max) values were almost the same whether the radiolabeled ligand was [^3H]isocarbacyclin (199 fmol/mg) or [^3H]iloprost (194 fmol/mg) in the NTS, although the value in the thalamus in the case of [^3H]iloprost (163 fmol/mg) was somewhat lower than that in the case of [^3H]isocarbacyclin (230 fmol/mg) (Fig. 3, A and B). This difference might be caused by the high concentration of [^3H]iloprost needed to saturate the binding because of the low affinity.


Figure 2: Difference in autoradiographic features between [^3H]iloprost and [^3H]isocarbacyclin binding in rat brain sections. Cryostat sections were incubated with 20 nM [^3H]iloprost (A) or 10 nM [^3H]isocarbacyclin (C) for 120 min at 4 °C, and the results were obtained as described under ``Experimental Procedures''. Nonspecific binding of [^3H]iloprost (B) and 10 nM [^3H]isocarbacyclin (D) is also shown. Arrows indicate the NTS, and arrowheads indicate the thalamus.




Figure 3: Scatchard plot analysis of isocarbacyclin (A) and iloprost (B) binding. The values of saturable specific [^3H]isocarbacyclin binding (1.5-90 nM) and saturable specific [^3H]iloprost binding (1.4-92 nM for the binding in the NTS and 20-430 nM for that in the thalamus) were transformed into the Scatchard plot. Scatchard plots for the NTS (box), thalamus (), and striatum (circle in A) are shown. B/F, bound/free.



Next, we examined the binding specificity using stable prostacyclin analogs and other PGs. Fig. 4A shows the prostacyclin analogs used in this study. Iloprost, cicaprost, and isocarbacyclin were reported to be potent agonists for the known prostacyclin receptor(20) . Carbacyclin was synthesized first as an agonist, but its agonistic potency was relatively weak(20) . 15R,16-(m-Tolyl)-17,18,19,20-tetranorisocarbacyclin (15R,16-(m-tolyl)isocarbacyclin) was a newly synthesized prostacyclin analog in our search for other prostacyclin derivatives with high affinity in the brain(25) . In the NTS, specific [^3H]isocarbacyclin binding was inhibited by unlabeled compounds in the order of isocarbacyclin = cicaprost = iloprost > carbacyclin > PGE(1) > 15R,16-(m-tolyl)isocarbacyclin > PGE(2) > PGD(2) = PGF (Fig. 4B). Note that among other prostaglandins, PGE(1) had relatively high affinity binding. In contrast, the binding property in the thalamus was apparently different, for specific [^3H]isocarbacyclin binding was inhibited in the order of isocarbacyclin = 15R,16-(m-tolyl)isocarbacyclin > carbacyclin > iloprost > PGE(2) > PGE(1) = cicaprost > PGD(2) = PGF (Fig. 4C). These results indicate that the prostacyclin receptor in the thalamus and most other brain regions is different from that expressed in the NTS.


Figure 4: A, chemical structures of prostacyclin analogs used in this study; B and C, displacement of [^3H]isocarbacyclin binding by various prostacyclin analogs and prostaglandins. Unlabeled compounds were added to the binding buffer at the indicated concentrations, and specific [^3H]isocarbacyclin binding in the NTS (B) or in the thalamus (C) was plotted. Prostacyclin analogs used were as follows: isocarbacyclin (bullet), iloprost (box), cicaprost (), carbacyclin (circle), and 15R,16-(m-tolyl)isocarbacyclin (). Prostaglandins used included PGE(1) (up triangle), PGE(2) (), PGD(2) (times), and PGF (down triangle). The results shown are representative of at least three similar experiments.



Then we examined the localization of the binding sites for [^3H]isocarbacyclin (Fig. 5). High density binding of this radiolabel was observed in the thalamus, lateral septal nucleus, hippocampus, cerebral cortex, striatum, and dorsal cochlear nucleus. These binding sites were clearly distinguished from the high density binding in the NTS and spinal trigeminal nucleus by the observations that 1) the former sites showed low density binding when [^3H]iloprost was used as a ligand for the mapping; and 2) the [^3H]isocarbacyclin binding in the former sites was unlikely to be displaced by cicaprost since cicaprost has very low affinity for the former sites. Little binding of [^3H]isocarbacyclin was observed in some regions, such as the cerebellum, substantia nigra, habenular nuclei, and medial septal nucleus.


Figure 5: In vitro autoradiographic localization of the binding sites for [^3H]isocarbacyclin. [^3H]Isocarbacyclin binding to rat brain coronal sections from rostral to caudal portions (from A to I) is shown. MOB, main olfactory bulb; CPu, caudate putamen; GP, globus pallidus; LSD, dorsal part of the lateral septal nucleus; VL, ventrolateral thalamic nucleus; HYP, hypothalamus; VPL, ventral posterolateral thalamic nucleus; CA1, field CA1 of Ammon's horn (hippocampus); DLG, dorsal lateral geniculate nucleus; SuG, superficial gray layer of the superior colliculus; MG, medial geniculate nucleus; DC, dorsal cochlear nucleus; Cer, cerebellum; Sp5C, caudal part of the spinal trigeminal nucleus.



To determine whether the binding sites are on neurons or afferents or other cells including glial cells, a hemilesion was made in the rat striatum with either kainate or 6-hydroxydopamine. Microinjections of kainate spare axon terminals of afferent neurons, but destroy the neuronal cell body in the striatum, while microinjections of 6-hydroxydopamine into the medial forebrain bundle destroy presynaptic terminals of the nigrostriatal tract in the striatum. Binding of the dopaminergic receptor and uptake site was used as a reference since the dopamine D(1) receptor is present on neuronal cells in the striatum, and dopamine uptake sites are found on presynaptic terminals of nigrostriatal afferent fibers. In vitro autoradiography revealed that [^3H]isocarbacyclin binding was markedly decreased by kainate treatment, whereas the binding was not affected by 6-hydroxydopamine treatment (Fig. 6). Both treatments induced a glial reaction (gliosis), as indicated by an increase in the content of glial fibrillary acid protein in the striatum (data not shown). These results suggest that the binding sites of [^3H]isocarbacyclin exist on neuronal cells in the striatum, but not on presynaptic terminals of afferents or on glial cells.


Figure 6: [^3H]Isocarbacyclin binding after hemilesioning of striatal neurons with kainate (A) or of afferents with 6-hydroxydopamine (B). Values of specific binding of [^3H]isocarbacyclin, [^3H]SCH23390, and [^3H]BTCP in the caudate putamen are shown. Closed bars, contralateral sides; open bars, ipsilateral sides.



The receptors for prostaglandins are known to be G protein-coupled receptors; and in most cases, ligand binding stimulates or inhibits adenylate cyclase activity. To test if this is also the case for the novel subtype of the prostacyclin receptor, we measured cAMP production in rat cortical slices after stimulation with isocarbacyclin in the concentration range of 10 to 10M. Increased cAMP production was not observed even at the highest concentration tested (66.4 ± 7.7 pmol/mg of protein at 10M isocarbacyclin; mean ± S.E., n = 6), where the control value was 109 ± 19.2 pmol/mg of protein. Isocarbacyclin also had no inhibitory effect on forskolin-stimulated cAMP production (239 ± 32.8 pmol/mg of protein at 10M isocarbacyclin versus 208 ± 41.8 pmol/mg of protein for the control). We also examined the effects of prostacyclin analogs on phosphoinositide turnover and calcium level in cortical slices and primary cultures of cortical neurons, respectively; but both experiments failed to demonstrate significant responses (data not shown). These results suggest that the signaling pathway downstream of this receptor is also different from that of the known prostacyclin receptor.

To assess the effect of prostacyclin analogs on neurons directly, we employed an electrophysiological recording in the CA1 region of the hippocampus since a high density binding of [^3H]isocarbacyclin was observed along the pyramidal cell layer. The application of isocarbacyclin caused a reversible enhancement of excitatory transmission in the region (Fig. 7A). Application of higher concentrations of isocarbacyclin (up to 10M) caused no greater enhancement than that of 10M (data not shown). This phenomenon was also observed by the application of prostacyclin itself, although the enhancement was not quantitatively estimated because of its short life. Carbacyclin, which has relatively low affinity for the known prostacyclin receptor, had a similar effect as isocarbacyclin, whereas cicaprost had significantly less potency than isocarbacyclin and carbacyclin at both concentrations tested (Fig. 7, B-D). These different potencies of prostacyclin analogs were comparable to binding affinities of these compounds for the novel subtype of the prostacyclin receptor, suggesting that the enhancement of excitatory transmission was caused by activation of the novel subtype.


Figure 7: Prostacyclin analogs enhance excitatory synaptic transmission in the hippocampus. Emsemble average of the EPSP slope (mean ± S.E., n = four slices from three to four rats) was plotted against time. After a 30-min base-line recording, prostacyclin analogs (box, 0.3 µM; bullet, 1 µM) were applied to the bath during the periods indicated (A, isocarbacyclin; B, carbacyclin; C, cicaprost). Three representative field EPSPs from experiments using 1 µM prostacyclin analogs are shown for the time points of 15, 45, and 75 min above each plot (A-C). A summary graph showing different responses by prostacyclin analogs at each concentration is shown in D. Average values between 41 and 50 min were used for the comparison (closed bars, isocarbacyclin; hatched bars, carbacyclin; open bars, cicaprost). Asterisks indicate statistical significance at the p < 0.05 level (Mann-Whitney test).




DISCUSSION

In this study, we identified and characterized a novel subtype of the prostacyclin receptor. The receptor had high affinity for a different set of prostacyclin analogs than the known prostacyclin receptor (IP)(26, 27) . Scatchard plot analysis showed a single high affinity site for isocarbacyclin, a prostacyclin analog. Iloprost, another prostacyclin analog, which is known to have high affinity for IP(26) , showed low affinity for the novel receptor. Other prostaglandins had no or little affinity for the subtype. Although PGE(1) has relatively high affinity for IP(26, 27) , it showed quite low affinity for the subtype. Distribution of [^3H]isocarbacyclin binding was also different from that of [^3H]PGD(2), [^3H]PGF, [^3H]PGE(1), and [^3H]PGE(2)(28, 29, 30, 31, 32) . Electrophysiological studies carried out in the hippocampus revealed that prostacyclin analogs enhance excitatory neural transmission, depending on a manner that the potencies of analogs correspond to their affinities for the novel subtype. From these results, the high affinity binding of [^3H]isocarbacyclin characterized in this study indicates the existence of a novel subtype of the prostacyclin receptor. We here propose to designate the known prostacyclin receptor as IP(1) and the novel subtype as IP(2).

The binding specificity of the prostacyclin receptor in the NTS was similar to that of IP(1), which was examined in the P815 mastocytoma cell line (26) and in Chinese hamster ovary cells transfected with cloned IP(1) cDNA(27) . The dissociation constant of 6.8 nM for [^3H]iloprost in the NTS was comparable to the value of 10.4 nM reported in P815 cells(26) . In the Scatchard plot analysis, no high affinity site for iloprost was expressed in the thalamus. Most of the other brain regions, except for the NTS and spinal trigeminal nucleus, showed binding properties similar to those of the thalamus. According to our designation, the receptor in the NTS and trigeminal nucleus is supposedly IP(1), and that in the other brain regions, IP(2). Intriguingly, the binding in the NTS was reported to be carried from nodose ganglion cells, which means the receptor in the NTS originates from the peripheral nervous system(19) . The fact that expression of IP(1) mRNA was not detected in mouse and human brains (27, 33) supports the idea that [^3H]isocarbacyclin-binding sites in the central nervous system represent IP(2) and also suggests that the novel subtype is structurally different from IP(1). E series prostaglandins also have structurally different receptor subtypes: EP(1), EP(2), and EP(3), which have overlapping ligand specificity(34, 35, 36) .

We also found that 15R,16-(m-tolyl)isocarbacyclin was a specific ligand for IP(2). 15R,16-(m-Tolyl)isocarbacyclin had high affinity for IP(2), but low affinity for IP(1). In agreement with results of the binding experiments, it showed a very weak inhibitory effect on platelet aggregation, (^2)whereas isocarbacyclin itself is a potent inhibitor of platelet aggregation. In this and other series of experiments, we found the carbon chain length and some features of the -chain to be critical in the binding of IP(2). It should be noted that carbacyclin, which has an alpha-chain identical to that of iloprost, but showed lower affinity for IP(1) than iloprost, exhibited high affinity for IP(2). 15S,16-(m-Tolyl)isocarbacyclin also had high affinity for IP(2), but it had relatively high affinity for IP(1) as well, resulting in little stereoselectivity at C-15. The RS configuration at C-15 thus appears not to be an important determinant for the binding of IP(2), although it seems to be important for the receptor binding of IP(1).^2

At present, all of the structurally identified prostanoid receptors are G protein-coupled receptors. For example, IP(1) is mainly coupled to G(s); and hence, the stimulation of the receptor results in cAMP production(27) . IP(2) had, however, no stimulatory effect on cAMP production and no inhibitory effect on forskolin-stimulated cAMP production. We also observed that prostacyclin analogs had no significant effects on phosphoinositide turnover and calcium level in cortical slices and primary cultures of cortical neurons, respectively. In our search for the function of prostacyclin in the central nervous system, however, we have found that isocarbacyclin causes a dose-dependent enhancement of the induced postsynaptic potential in the CA1 region of the hippocampus at concentrations under 10M. Further analysis confirmed that the site of action is excitatory synapses between CA3 and CA1 pyramidal neurons. (^3)Prostacyclin showed a similar response as isocarbacyclin; but 6-keto-PGF, a degradation product of prostacyclin, did not.^3 These results suggest that prostacyclin can elicit a receptor-mediated response from neurons and that isocarbacyclin is an agonist for IP(2). This subtype might be coupled with some effector pathway other than that involving adenylate cyclase and calcium mobilization(37) . Alternatively, there is a possibility that the sensitivity of second messenger analysis of our system is not enough to detect subtle change of the messenger without a cell line that abundantly expresses the receptor. This issue should be further analyzed in the future through cDNA isolation-expression experiments or pharmacological intervention in hippocampal electrophysiological experiments using protein kinase inhibitors and stimulators of second messenger systems.

There are few other studies that have investigated the function of prostacyclin in the central nervous system. It seems that the very short half-life of prostacyclin and lack of stable analogs specific for IP(2) have hampered experimental and clinical investigations. Clinically, there are many prostacyclin analogs being assessed for medicinal use, mostly for the purpose of regulation of blood coagulation and vascular tone. Of these, TEI9090, a methyl ester of isocarbacyclin, was reported to be effective in improving the neurological symptoms of patients suffering from the aftereffects of ischemic cerebrovascular attack(38) . Although the precise mechanisms remain to be elucidated, the widespread expression of this newly found prostacyclin receptor subtype in the central nervous system suggests that prostacyclin plays some important roles in neuronal activity.


FOOTNOTES

*
This work was supported in part by the special coordination funds for promoting science and technology from the Science and Technology Agency of Japan and by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Neuroscience, Osaka Bioscience Inst., 6-2-4 Furuedai, Suita-shi, Osaka 565, Japan. Tel.: 81-6-872-4852; Fax: 81-6-872-0240.

(^1)
The abbreviations used are: PGs, prostaglandins; NTS, nucleus of the solitary tract; BTCP, N-[1-(2-benzo(b)thiophenyl)cyclohexyl] piperidine; EPSP, excitatory postsynaptic potential; 15R,16-(m-tolyl)isocarbacyclin, 15R,16-(m-tolyl)-17,18,19,20-tetranorisocarbacyclin; IP, prostacyclin receptor; EP, prostaglandin E receptor.

(^2)
Y. Watanabe, H. Takechi, K. Matsumura, K. Kato, R. Noyori, M. Suzuki, and Y. Watanabe, unpublished observation.

(^3)
H. Takechi, K. Matsumura, and Y. Watanabe, unpublished observation.


ACKNOWLEDGEMENTS

We thank Dr. Seizi Kurozumi and Dr. Atsuo Hazato (Teijin Ltd.) for helpful discussion and for providing [^3H]isocarbacyclin and isocarbacyclin (TEI7165). We also thank Dr. Toshiya Manabe (Tokyo University) for helpful comments on our electrophysiological experiments.


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