(Received for publication, June 9, 1995; and in revised form, November 27, 1995)
From the
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.
[
H]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 [
H]isocarbacyclin binding,
[
H]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.
Prostaglandins (PGs) ()and thromboxane are formed
from arachidonic acid by cyclooxygenase and the respective synthase for
each PG, such as PGD synthase and PGI
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
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
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
[H]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
[H]isocarbacyclin was preliminarily used in the
autoradiographic study instead of [
H]iloprost, we
found that its binding potency was much higher than that of
[
H]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 [
H]isocarbacyclin-binding site
in the rat brain with special attention to its distinction from those
of the [
H]iloprost-binding site. We demonstrated
thereby that most of the prostacyclin receptors in the brain are
distinct from the previously known prostacyclin receptor.
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
[H]isocarbacyclin binding in the cryostat
sections of the rat brain. At 25 °C, the specific binding of
[
H]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
[
H]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 [H]isocarbacyclin binding. Cryostat sections
were incubated with 10 nM [
H]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 [
H]isocarbacyclin. Total (
,
),
specific (
,
), and nonspecific (
,
) binding
and dissociation (
) 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) , [H]iloprost binding in the
NTS was much higher than that in the thalamus when 20 nM [
H] iloprost was used, two times higher than
the known K
value (Fig. 2A). The
binding study using 10 nM [
H]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 [
H]isocarbacyclin and
[
H]iloprost, we performed Scatchard plot
analysis. Scatchard analysis carried out using
[
H]isocarbacyclin showed single high affinity
sites in the NTS, thalamus, and striatum, with K
values of 3.9, 7.8, and 8.9 nM, respectively (Fig. 3A). K
values for both the
thalamus and striatum were thus slightly higher than the value for the
NTS. The difference was much more evident when
[
H]iloprost was used as a ligand (Fig. 3B). For the NTS, the K
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 [
H]iloprost were not observed in the thalamus (Fig. 3B). B
values were almost
the same whether the radiolabeled ligand was
[
H]isocarbacyclin (199 fmol/mg) or
[
H]iloprost (194 fmol/mg) in the NTS, although
the value in the thalamus in the case of
[
H]iloprost (163 fmol/mg) was somewhat lower than
that in the case of [
H]isocarbacyclin (230
fmol/mg) (Fig. 3, A and B). This difference
might be caused by the high concentration of
[
H]iloprost needed to saturate the binding
because of the low affinity.
Figure 2:
Difference in autoradiographic features
between [H]iloprost and
[
H]isocarbacyclin binding in rat brain sections.
Cryostat sections were incubated with 20 nM [
H]iloprost (A) or 10 nM [
H]isocarbacyclin (C) for 120 min
at 4 °C, and the results were obtained as described under
``Experimental Procedures''. Nonspecific binding of
[
H]iloprost (B) and 10 nM [
H]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 [H]isocarbacyclin binding (1.5-90
nM) and saturable specific [
H]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 (
),
thalamus (
), and striatum (
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 [H]isocarbacyclin binding was inhibited
by unlabeled compounds in the order of isocarbacyclin =
cicaprost = iloprost > carbacyclin > PGE
>
15R,16-(m-tolyl)isocarbacyclin > PGE
> PGD
= PGF
(Fig. 4B). Note that among other prostaglandins,
PGE
had relatively high affinity binding. In contrast, the
binding property in the thalamus was apparently different, for specific
[
H]isocarbacyclin binding was inhibited in the
order of isocarbacyclin =
15R,16-(m-tolyl)isocarbacyclin > carbacyclin >
iloprost > PGE
> PGE
= cicaprost
> PGD
= 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
[H]isocarbacyclin binding by various prostacyclin
analogs and prostaglandins. Unlabeled compounds were added to the
binding buffer at the indicated concentrations, and specific
[
H]isocarbacyclin binding in the NTS (B)
or in the thalamus (C) was plotted. Prostacyclin analogs used
were as follows: isocarbacyclin (
), iloprost (
),
cicaprost (
), carbacyclin (
), and
15R,16-(m-tolyl)isocarbacyclin (
).
Prostaglandins used included PGE
(
), PGE
(
), PGD
(
), and PGF
(
). The results shown are representative of at least three
similar experiments.
Then we examined the
localization of the binding sites for
[H]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
[
H]iloprost was used as a ligand for the mapping;
and 2) the [
H]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
[
H]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
[H]isocarbacyclin.
[
H]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 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
[
H]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
[
H]isocarbacyclin exist on neuronal cells in the
striatum, but not on presynaptic terminals of afferents or on glial
cells.
Figure 6:
[H]Isocarbacyclin
binding after hemilesioning of striatal neurons with kainate (A) or of afferents with 6-hydroxydopamine (B).
Values of specific binding of [
H]isocarbacyclin,
[
H]SCH23390, and [
H]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
10
M. Increased cAMP production was not
observed even at the highest concentration tested (66.4 ± 7.7
pmol/mg of protein at 10
M 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 10
M 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
[H]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 10
M) caused no
greater enhancement than that of 10
M (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 (, 0.3 µM;
,
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).
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 has relatively high affinity for
IP(26, 27) , it showed quite low affinity for the
subtype. Distribution of [
H]isocarbacyclin
binding was also different from that of
[
H]PGD
,
[
H]PGF
,
[
H]PGE
, and
[
H]PGE
(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 [
H]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
and the novel subtype as
IP
.
The binding specificity of the prostacyclin receptor
in the NTS was similar to that of IP, which was examined in
the P815 mastocytoma cell line (26) and in Chinese hamster
ovary cells transfected with cloned IP
cDNA(27) .
The dissociation constant of 6.8 nM for
[
H]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
, and that in the other brain regions, IP
.
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
mRNA was not detected in mouse and human
brains (27, 33) supports the idea that
[
H]isocarbacyclin-binding sites in the central
nervous system represent IP
and also suggests that the
novel subtype is structurally different from IP
. E series
prostaglandins also have structurally different receptor subtypes:
EP
, EP
, and EP
, which have
overlapping ligand specificity(34, 35, 36) .
We also found that 15R,16-(m-tolyl)isocarbacyclin
was a specific ligand for IP.
15R,16-(m-Tolyl)isocarbacyclin had high affinity for
IP
, but low affinity for IP
. In agreement with
results of the binding experiments, it showed a very weak inhibitory
effect on platelet aggregation, (
)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
. It should be noted that carbacyclin, which has an
-chain identical to that of iloprost, but showed lower affinity
for IP
than iloprost, exhibited high affinity for
IP
. 15S,16-(m-Tolyl)isocarbacyclin also
had high affinity for IP
, but it had relatively high
affinity for IP
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
, although it seems to be important for the receptor
binding of IP
.
At present, all of the
structurally identified prostanoid receptors are G protein-coupled
receptors. For example, IP is mainly coupled to
G
; and hence, the stimulation of the receptor results in
cAMP production(27) . IP
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 10
M. Further analysis
confirmed that the site of action is excitatory synapses between CA3
and CA1 pyramidal neurons. (
)Prostacyclin showed a similar
response as isocarbacyclin; but 6-keto-PGF
, a
degradation product of prostacyclin, did not.
These results
suggest that prostacyclin can elicit a receptor-mediated response from
neurons and that isocarbacyclin is an agonist for IP
. 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 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.