Yumiko Watanabe

Mapping of prostaglandin E2 binding sites in rat brain using quantitative autoradiography

The density of specific prostaglandin E2 (PGE2) binding sites was quantitatively mapped in the rat brain using in vitro autoradiography. The anterior wall of the third ventricle and the nucleus solitary tract were found to have a very high density of binding sites (greater than 15 fmol/mg tissue). Two thalamic nuclei (paraventricular and anteroventral nuclei) and the dorsal parabrachial nucleus contained a high density of binding sites (10-15 fmol/mg tissue). Entorhinal cortex, ventral hippocampus, amygdala, dorsomedial hypothalamus, mammillary complex, some thalamic nuclei, central gray, superior colliculus, raphe nuclei, locus coeruleus, spinal trigeminal nucleus (caudal part) and the dorsal horn of the spinal cord (laminae 1 and 2) had each a moderate density of binding sites (5-10 fmol/mg tissue). Binding tended to occur in brain regions rich in neuronal cell bodies or neuronal cell processes (dendrites and axon terminals). PGE1, whose central actions are very similar to those of PGE2, had essentially the same pattern of binding sites as did PGE2 throughout the entire brain, suggesting there are receptors common to these two PGEs. In addition to already known functions of receptors common to these two PGEs. In addition to already known functions of PGE2 in the hypothalamus, which include fever genesis, promotion of wakefulness, cardiovascular control and LH-RH release, the unique distribution of extrahypothalamic PGE2 binding sites found in this study suggests its involvement in the processing or modulation of viscerosensory, somatosensory (nociceptive and possibly thermal) and visual inputs as well as in the central integration of autonomic and limbic functions.(ABSTRACT TRUNCATED AT 250 WORDS)

High density of prostaglandin E2 binding sites in the anterior wall of the 3rd ventricle: a possible site of its hyperthermic action

Prostaglandin E2 (PGE2) exerts a potent hyperthermic action when injected into the preoptic-hypothalamic area (POHA) and is considered to be a central mediator of fever. To determine the exact functional sites of PGE2, we used in vitro quantitative autoradiography of [3H]PGE2 binding sites in the rat POHA. The highest density of [3H]PGE2 binding was found in the regions of the anterior wall of the 3rd ventricle (A3V). Within the A3V, binding density was especially high in regions closest to the third ventricle or surrounding the organum vasculosum laminae terminalis (OVLT) but was relatively low within the OVLT itself. It seems likely that the A3V PGE2 binding sites identified in this study are responsible for PGE2 mediation of fever.

Prostacyclin receptor in the brain and central terminals of the primary sensory neurons: An autoradiographic study using a stable prostacyclin analogue [3H]iloprost

Presence and localization of the prostacyclin receptor in the rat brain and spinal cord were examined by in vitro autoradiography using [3H]iloprost, a highly specific and stable agonist for this receptor. Density of specific binding sites for iloprost was generally high in four regions of the lower brain stem, that is, the medial and commissural subnuclei of the nucleus tractus solitarius, the area postrema, superficial layers of the spinal trigeminal nucleus caudalis and dorsal horn. Moderate density was found in the thalamus, cerebral cortex, hippocampus, striatum and dorsal cochlear nucleus. The distribution pattern was distinct from those of other prostanoid binding sites previously studied except that prostaglandin E2 binding sites were also abundant in the nucleus tractus solitarius, spinal trigeminal nucleus caudalis and dorsal horn. Even in these regions, binding sites for iloprost had several features clearly different from those for prostaglandin E2. First, within the medial and commissural subnuclei of the nucleus tractus solitarius, iloprost binding sites were distributed preferentially in the dorsal part, while those for prostaglandin E2 were located more ventrolaterally. Second, on postnatal day 0, iloprost binding sites have already been expressed in large amounts in the nucleus tractus solitarius, spinal trigeminal nucleus caudalis and dorsal horn of rats, while prostaglandin E2 binding sites are negligible at this stage. Thirdly, the binding of 10 nM [3H]iloprost in these three regions was almost completely displaced by 10 microM unlabelled iloprost but only slightly by 10 microM unlabelled prostaglandin E2. Unilateral nodose ganglionectomy or dorsal rhizotomy decreased the density of iloprost binding sites in the nucleus tractus solitarius or dorsal horn, respectively, with a greater decrease in the operated side. Ligation of the vagus either central or peripheral to the nodose ganglion resulted in an accumulation of iloprost binding sites proximal to the ligation. These results suggest that specific binding sites for iloprost, presumably prostacyclin receptor, are present in the nervous system and, in particular, that the iloprost binding sites in the nucleus tractus solitarius, dorsal horn and possibly in the superficial layers of the spinal trigeminal nucleus caudalis are produced in their sensory ganglia and transported to central terminals of the primary sensory afferents as well as to their peripheral terminals.

Localization of cytosolic phospholipase A2 messenger RNA mainly in neurons in the rat brain

Ca2(+)-sensitive 85,000 mol. wt cytosolic phospholipase A2 plays an essential role in the selective and stimulus-dependent release of arachidonic acid from membrane phospholipids. Cytosolic phospholipase A2-catalysed lipid mediators including arachidonic acid and its metabolites have been suggested to be involved in a variety of neuronal functions in the CNS. Since the cellular localization of cytosolic phospholipase A2 is still controversial and obscure, we tried an improved method of rapid processing of each specimens and succeeded in obtaining intense signals of cytosolic phospholipase A2 messenger RNA in the normal rat brain by northern blot analysis and in situ hybridization. Northern blot analysis showed the abundant distribution of cytosolic phospholipase A2 messenger RNA in most regions of the brain, with intense signals observed in the pineal gland and pons. Macroautoradiographs prepared after in situ hybridization with three different antisense riboprobes gave essentially similar patterns of localization; significant signals were widely detected in the gray matter of various regions, i.e. the olfactory bulb, cerebral cortex, hippocampus, amygdala, several thalamic and hypothalamic nuclei and cerebellum. Microautoradiographs showed that most of the intense signals were predominant in neurons, and that faint signals were from glial cells and other non-neuronal cells in the choroid plexus, inner surface cells of veins and the leptomeninges. In addition, the cycloheximide treatment increased the cytosolic phospholipase A2 messenger RNA level in the same cell populations originally possessing messenger RNA signals. Predominant expression of cytosolic phospholipase A2 messenger RNA in neurons may provide the basis for the contribution of cytosolic phospholipase A2-catalysed lipid mediators to a variety of neurotransmission and synaptic functions in the CNS.

A Novel Subtype of the Prostacyclin Receptor Expressed in the Central Nervous System

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.

Chapter 15 Prostaglandin system in the brain: sites of biosynthesis and sites of action under normal and hyperthermic states

Publisher Summary This chapter reviews the recent progress in research on the brain prostaglandin (PG) system, with special attention on the in vivo location of enzymes involved in their biosynthesis and of their specific receptors under physiological, as well as pathological conditions. The recent biochemical studies, especially those that employed molecular biological techniques, have provided a great deal of information on the molecular nature of individual components of the PG system. Fever and hyperthermia are essentially distinct patho-physiological states, the former representing regulated elevation of the body temperature caused by immunological challenge and the latter representing its passive elevation caused by excessive heat load. In fact, inhibitors of PG synthesis, such as indomethacin, suppress fever but are not effective against hyperthermia. The release of PGs is dramatically increased by ischemia/reperfusion of the brain or by traumatic injury of the central nervous and treatment with a cyclooxygenase inhibitor, such as indomethacin, improves the neuronal injury. The onset and progression of Alzheimers disease may be also slowed by the treatment with cyclooxygenase inhibitors. Thus, PGs are the key molecules involved in pathological changes in the central nervous system.

Differential expression of immediate-early genes, c-fos and zif268, in the visual cortex of young rats: effects of a noradrenergic neurotoxin on their expression.

We investigated the expression pattern of two immediate-early genes, zif268 and c-fos, under various visual conditions using immunohistochemical and northern blot analysis in the visual cortex of young rats. The basal expression of c-fos was low and was further reduced by dark rearing that lasted for one week. A marked and transient increase was induced upon visual stimulation applied immediately after dark rearing. Zif268 showed a relatively high basal level. Its expression was reduced by dark rearing of the animals, but returned rapidly to the basal expression level following the introduction of light. Administration of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, a selective noradrenergic neurotoxin, suppressed the basal expression of c-fos messenger RNA. The response of c-fos to photo-stimulation was also significantly lower in the visual cortex of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine-treated young rats. In contrast, no significant change in zif268 expression was detected between normal and N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine-treated animals. These findings suggest that differential expression of these immediate-early genes is involved in the activity-dependent regulation of cortical function. One possibility is that the noradrenergic system controls cortical function, including plasticity, by modifying the expression of c-fos.

CNS-specific prostacyclin ligands as neuronal survival-promoting factors in the brain

Prostacyclin (PGI2) is a critical regulator of the cardiovascular system, via dilatation of vascular smooth muscle and inhibition of platelet aggregation (Moncada, S. 1982, Br. J. Pharmacol., 76, 3). Our previous studies demonstrated that a novel subtype of PGI2 receptor, which is clearly distinct from a peripheral subtype in terms of ligand specificity, is expressed in the rostral region of the brain, e.g. cerebral cortex, hippocampus, thalamus and striatum, and that (15r)‐16‐m‐17,18,19,20‐tetranorisocarbacyclin (15r‐TIC) and 15‐deoxy‐16‐m‐17,18,19,20‐tetranorisocarbacyclin (15‐deoxy‐TIC) specifically bind to the central nervous system (CNS)‐specific PGI2 receptor. Here, we report that these CNS‐specific PGI2 receptor ligands, including PGI2 itself, prevented the neuronal death. They prevented apoptotic cell death of hippocampal neurons induced by high (50%) oxygen atmosphere, xanthineu2003+u2003xanthine oxidase, and serum deprivation. IC50s for neuronal death were ∼u200330 and 300u2003nm for 15‐deoxy‐TIC and 15r‐TIC, respectively, which well correlated with the binding potency for the CNS‐specific PGI2 receptor. 6‐Keto‐PGF1α (a stable metabolite of PGI2), peripheral nervous system‐specific PGI2 ligands and other prostaglandins (PGs) than PGI2 did not show such neuroprotective effects. In vivo, 15r‐TIC protected CA1 pyramidal neurons against ischaemic damage in gerbils. These results indicate that CNS‐specific PGI2 ligands have neuronal survival‐promoting activity both in vitro and in vivo, and may represent a new type of therapeutic drug for neurodegeneration.

Difference in response of D2 receptor binding between 11C-N-methylspiperone and 11C-raclopride against anesthetics in rhesus monkey brain

The effects of anesthesia on dopamine D2 receptor binding in the rhesus monkey brain were examined using positron emission tomography. The bindings of11C-N-methylspiperone (NMSP) and11C-raclopride (RAC) were measured under controlled ketamine or isoflurane anesthesia. The binding of11C-NMSP was significantly lower in the striatum anesthetized with isoflurane than with ketamine. There was a smaller change in the binding of11C-RAC than of11C-NMSP. These findings suggest that changes in11C-NMSP or11C-RAC binding induced by anesthetics were not due solely to changes in the competition of endogenous dopamine.

Specific ligand for a central type prostacyclin receptor attenuates neuronal damage in a rat model of focal cerebral ischemia.

The neuroprotective effect of a central type prostacyclin receptor ligand was examined in a rat model of focal cerebral ischemia. Under halothane anesthesia, male Sprague-Dawley rats were subjected to left middle cerebral artery occlusion. A selective central type prostacyclin receptor ligand, 15-deoxy-(16-m-tolyl)-17,18,19,20-tetranorisocarbacyclin methylester, or a peripheral type prostacyclin receptor ligand, iloprost methylester, were administered intravenously immediately after ischemia. Twenty-four hours after ischemia, brain damage was evaluated. In separate experiments, concentrations of 15-deoxy-(16-m-tolyl)-17,18,19,20-tetranorisocarbacyclin in ischemic brain tissue were measured by injection of a tritium labeled compound. Treatment with 15-deoxy-(16-m-tolyl)-17,18,19,20-tetranorisocarbacyclin methylester (0.03 mg/kg) significantly (P<0.05) reduced the volume of brain damage by 35%. With this treatment, the concentration of this compound in the brain was more than 10 nM. Treatment with iloprost methylester did not show a neuroprotective effect. These results indicated that activation of a central type prostacyclin receptor attenuates ischemic brain damage. The present study demonstrated that the intravenous application of a central type prostacyclin receptor ligand could be a novel therapeutic agent for acute stroke.