Seishi Katsumata

In situ hybridization study of interleukin-1β mRNA induced by kainic acid in the rat brain

The distribution patterns of interleukin-1 beta (IL-1 beta) mRNA in various brain regions of saline- and kainic acid-treated rats were examined using in situ hybridization technique. In normal rat brain, the signals of IL-1 beta mRNA were observed in the cerebellar Purkinje cells and in dispersed cells in the hypothalamus. In the case of the kainic acid treatment, IL-1 beta mRNA was intensely induced in the olfactory bulb, lateral septum, thalamus, hypothalamus, polymorphic layers of hippocampus, piriform cortex, amygdala, entorhinal cortex and cerebral cortex at 2 h after the injection of kainic acid. In the hypothalamic region, we observed the induction of IL-1 beta mRNA around the paraventricular hypothalamic nucleus, anterior hypothalamic area, dorsomedial and ventromedial hypothalamic nucleus, mammillary regions and arcuate nucleus. The signal of IL-1 beta mRNA was still expressed 4 h after treatment with kainic acid, less intensely than at 2 h, but above the control level. In these regions, IL-1 beta mRNA was expressed mainly in the glial cells, which were densely stained by Cresyl violet and did not contain glial fibrillary acidic protein. These results suggest that IL-1 beta is produced by a certain type of glial cells, maybe microglia, and might have regulatory functions in the central nervous system.

An in situ hybridization study on interleukin-1β mRNA induced by transient forebrain ischemia in the rat brain

Expression of interleukin-1 beta (IL-1 beta) mRNA in the rat brain after transient forebrain ischemia was investigated by in situ hybridization histochemistry. Thirty min after the start of recirculation, IL-1 beta mRNA was induced in the several brain regions, including the olfactory bulb, cerebral cortex, hippocampus, striatum and thalamus where neuronal degeneration was reported to be observed after transient forebrain ischemia. The hybridization signals were observed both on the glial cells and around the vascular walls.

Molecular cloning and in situ hybridization histochemistry for rat μ-opioid receptor

Abstract We cloned a cDNA for the rat μ-opioid receptor from a rat thalamus cDNA library. The deduced amino-acid sequence of rat μ-opioid receptor consists of 398 residues with the features shared by the members of the G-protein coupled receptor family, and is 59% and 60% identical with those of rat κ-opioid and mouse δ-opioid receptors, respectively. Northern blot analysis showed that expression of μ-opioid receptor mRNA was intensive in the thalamus, striatum, hypothalamus and pons-medulla, moderate in the hippocampus and midbrain, and slight in the cerebral cortex and cerebellum. More detailed distribution of the mRNA in the rat brain was examined using the in situ hybridization technique. Intense expression of μ-opioid receptor mRNA was observed in the internal granular and glomerular layers of the olfactory bulb, caudate putamen, nucleus accumbens, medial raphe nucleus, inferior colliculus, parabrachial nucleus, locus coeruleus, nucleus solitary tract and ambiguus nucleus. Furthermore, μ-opioid receptor mRNA was moderately expressed in the hippocampus, globus pallidus, ventral pallidus, arcuate hypothalamic nucleus, supramammillary nucleus, superior colliculus, periacqueductal gray, and several nuclei of lower brain stem, including raphe magnus nucleus, reticular gigantocellular nucleus and lateral paragigantocellular nucleus.

DAMGO, a μ-opioid receptor selective agonist, distinguishes between μ- and δ-opioid receptors around their first extracellular loops

The structural basis of μ‐opioid receptor (OPR) for the specificity in its ligand binding was investigated using chimeric μ/δ‐OPRs. Replacement of the region around the first extracellular loop of δ‐OPR with the corresponding region of μ‐OPR gave the resultant chimeric receptor the similar affinity to DAMGO compared with the native μ‐OPR. The reciprocal replacement deprived the high affinity to DAMGO from μ‐OPR. These results indicate that the difference(s) in the structure around the first extracellular loop is critical for DAMGO to distinguish between μ‐ and δ‐OPRs. Furthermore, displacement studies revealed that this region is partly involved in the discrimination between μ‐ and δ‐OPRs by other peptidic μ‐selective ligands, such as dermorphin, morphiceptin and CTOP, but not by non‐peptidic ligands, such as morphine and naloxone.

DAMGO, a μ‐opioid receptor selective ligand, distinguishes between μ‐and κ‐opioid receptors at a different region from that for the distinction between μ‐ and δ‐opioid receptors

The structural basis of opioid receptors (OPRs) for the subtype‐selective binding of DAMGO, a μ‐opioid receptor selective ligand, was investigated using chimeric μ/κ‐OPRs. Replacement of the region from the middle of the fifth transmembrane domain to the C‐terminal of μ‐OPR with the corresponding region of μ‐OPR remarkably decreased the binding affinity to DAMGO, while the reciprocal chimera revealed the high affinity to DAMGO. These results indicate that DAMGO distinguishes between μ‐ and μ‐OPRs at the region around the third extracellular loop, different from the case of the distinction between μ‐and δ‐OPRs in which the region around the first extracellular loop is important. Furthermore, displacement studies revealed that the region around the third extracellular loop is involved in the discrimination between μ‐ and κ‐OPRs not only by peptidic μ‐ selective ligands but also by non‐peptidic ligands, such as morphine and naloxone.

In situ hybridization study of κ-opioid receptor mRNA in the rat brain

Abstract Distribution of κ-opioid receptor mRNA in rat brain was examined by in situ hybridization technique. κ-Opioid receptor mRNA was expressed in various brain regions, especially intensely in the neocortex (layer V and VI), caudate-putamen, nucleus accumbens, preoptic area, paraventricular thalamic nucleus, amygdala, several nuclei of hypothalamus, ventral tegmental area and substantia nigra pars compacta.

Pharmacological study of dihydroetorphine in cloned μ-, δ- and κ-opioid receptors

Abstract We investigated the binding characteristics of dihydroetorphine, 7,8-dihydro-7α-[1-(R)-hydroxyl-1-methylbutyl]-6,14-endoethano-tetrahydro-oripavine, and its effect on the inhibitory system of cyclic AMP production using cloned μ-, δ- and κ-opioid receptors expressed on Chinese hamster ovary cells. The Ki values of dihydroetrophine for the μ-, δ- and κ-opioid receptor were 4.5 × 10−10, 1.8 × 10−9 and 5.7 × 10−10 M, respectively. On the hand, those of morphine were 1.9 × 10−9, 1.4 × 10−6 and 1.3 × 10−7 M, respectively. Through all of these three types of opioid receptors, dihydroetorphine inhibited forskolin (10 μM)-stimulated cyclic AMP production via pertussis toxin-sensitive G protein(s), and the inhibitory effects were antagonized by co-application with opioid receptor antagonists. The IC50 values of dihydroetorphine for the inhibition of cyclic AMP production through the μ-, δ- and κ-opioid receptors were 4.2 × 10−11, 8.6 × 10−10 and 4.3 × 10−9 M, respectively. On the other hand, those of morphine were 2.6 × 10−8, 2.6 × 10−6 and 1.9 × 10−6 M, respectively. These results indicate that dihydroetorphine, unlike morphine which preferentially binds the μ-opioid receptor, binds not only μ- but also δ- and κ-opioid receptors with high affinity and acts as a more potent agonist than morphine for all of the three types of receptors.

Suppression of naloxone‐precipitated withdrawal jumps in morphine‐dependent mice by stimulation of prostaglandin EP3 receptor

1 . We have shown that intracisternal (i.c.) administration of interleukin‐1β (IL‐1β) attenuates naloxone‐precipitated withdrawal jumps in morphine‐dependent mice, and the effect was partly mediated by the corticotropin‐releasing factor. To elucidate further other possible mechanisms involved in the inhibitory effect of IL‐1β on morphine withdrawal jumping behaviour, in this study, we examined the involvement of the prostaglandin‐synthesis pathway, because prostaglandins have been shown to mediate the several central effects of IL‐1. Furthermore, we examined the effects of subtype‐selective prostaglandin receptor agonists on morphine withdrawal jumping behaviour. 2 . Mice were rendered morphine‐dependent by subcutaneous implantation of a pellet containing 11.5±0.3 mg morphine hydrochloride for 48 h. Morphine withdrawal syndromes were precipitated by intraperitoneal (i.p.) injection of naloxone (10 mg kg−1). The degree of physical dependence on morphine was estimated by counting the number of jumps, one of the typical withdrawal signs in mice, for 40 min. 3 . The inhibitory effect of IL‐1β (1 ng/mouse) administered intracisternally 30 min before naloxone (10 mg kg−1, i.p.) was significantly blocked by pretreatment with sodium salicylate (a cyclo‐oxygenase inhibitor, 10 ng or 30 ng/mouse) administered intracisternally 15 min before IL‐1β, while i.c. administration of sodium salicylate alone (3 ng, 10 ng or 30 ng/mouse) followed by i.c. administration of vehicle instead of IL‐1)? did not significantly change the number of jumps precipitated by naloxone. 4 . Intracisternal administration of M&B28,767 (an EP3‐receptor agonist, 1 fg‐ 30 ng/mouse) and sulprostone (an EP1/EP3‐receptor agonist, 10 fg‐100 ng/mouse) 30 min before naloxone (10 mg kg−1 i.p.) attenuated withdrawal jumps with a U‐shaped dose‐response, reaching a peak at 10 pg/mouse and 100 pg/mouse, respectively. On the other hand, i.c. administration of iloprost (an EP1/IP‐receptor agonist, 10fg‐100 ng/mouse), butaprost (an EP2‐receptor agonist, 10 fg‐100 ng/mouse) or prostaglandin F2α (a FP‐receptor agonist, 10 fg‐100 ng/mouse) 30 min before naloxone (10 mg kg−1, i.p.) did not significantly change the number of jumps precipitated by naloxone. 5 . These results indicate that the prostaglandin‐synthesis pathway is, at least in part, involved in the inhibitory effect of IL‐1β on naloxone‐precipitated withdrawal jumps in morphine‐dependent mice, and that the prostaglandin synthesized in the brain suppresses the morphine withdrawal jumping behaviour via the EP3‐receptor, but not via the EP1, EP2‐, IP‐ or FP‐receptor.

Intracisternal administration of interleukin-1β attenuates naloxone-precipitated withdrawal in morphine-dependent mice

The effect of central administration of interleukin-1 beta on naloxone-precipitated withdrawal in morphine-dependent mice was studied. The degree of physical dependence on morphine was estimated by counting the number of jumps precipitated by naloxone, one of the typical withdrawal signs. Intracisternal (i.c.) administration of interleukin-1 beta (0.01-1 ng/5 microliters per mouse) to morphine-dependent mice 30 min prior to the injection of naloxone (10 mg/kg i.p.) decreased the number of jumps in a dose-dependent manner. The effect of interleukin-1 beta (1 ng) was significantly antagonized when it was co-administered with interleukin-1 receptor antagonist (1 microgram/mouse). These results suggest that centrally administered interleukin-1 beta could attenuate naloxone-precipitated withdrawal in morphine-dependent mice via interleukin-1 receptors in the brain. Co-administration of alpha-melanocyte-stimulating hormone (300 ng/mouse) or alpha-helical corticotropin-releasing factor (CRF)-(9-41), a CRF receptor antagonist (300 ng/mouse), with interleukin-1 beta also antagonized the inhibitory effect of interleukin-1 beta (1 ng). Moreover, i.c. administration of CRF (200 ng/mouse) significantly decreased the number of jumps.

Pharmacological characterization of KT-90 using cloned μ-, δ- and κ-opioid receptors

Abstract We analyzed the pharmacological characteristics of (−)-3-acetyl-6β-acetylthio-N-cyclopropylmethyl-normorphine (KT-90) using Chinese hamster ovary (CHO) cells expressing cloned rat μ-, δ- and κ-opioid receptors. KT-90 displaced the specific binding of the following radiolabeled ligands selective to the μ-, δ- and κ-opioid receptors, [3H][ d -Ala2,MePhe4,Gly(ol)5]enkephalin (DAMGO), [3H][ d -Pen2, d -Pen5]enkephalin (DPDPE), [3H] (+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro-4,5)dec-8-yl]benzeneacetamide (U69,593), with Ki values of 3.3 ± 0.7, 22.8 ± 1.5 and 1.9 ± 0.3 nM, respectively. In CHO cells expressing the μ-, δ- and κ-opioid receptors, KT-90 inhibited forskolin (10 μM)-induced cyclic AMP accumulation in a concentration-dependent manner with IC50 values of 2337 ± 750, 17.3 ± 4.6 and 2.0 ± 0.1 nM, respectively. The maximal inhibitory effects of KT-90 in the cells expressing μ-, δ- and κ-opioid receptors were significantly lower than those of the type-selective agonists DAMGO, DPDPE and U69,593, respectively. These results indicated that KT-90 acts as a partial agonist on μ-, δ and κ-opioid receptors. KT-90 (10 and 100 nM), when added with morphine, produced a rightward shift of the concentration-response curve of morphine to inhibit the cyclic AMP accumulation in CHO cells expressing μ-, but not δ- or κ-, opioid receptors. This finding is consistent with the findings that lower doses of KT-90 antagonize morphine analgesia in vivo.