Keiko Maekawa

Cloning and expression of a cDNA for the rat k-opioid receptor

We cloned a cDNA for the rat K‐opioid receptor from a rat thalamus cDNA library. The deduced amino acid sequence consists of 380 residues with features shared by members of the G protein‐coupled receptor family. The specific binding of [3H]bremazocine to the membrane of COS‐7 cells transfected with the cDNA was displaced by k‐specific opioid ligands, but not by μ‐ and δ‐specific ligands. Xenopus oocytes injected with the in vitro transcribed mRNA responded to opioid ligands with the same subtype specificity. Northern blot analysis demonstrated that k‐opioid receptor mRNA is expressed in a regionally specific manner in rat brain.

Double in situ hybridization study on coexistence of μ-, δ- and κ-opioid receptor mRNAs with preprotachykinin A mRNA in the rat dorsal root ganglia

Coexistence of the mRNA for each subtype of opioid receptor (OPR) with the mRNA for preprotachykinin A (PPTA), a precursor protein of substance P (SP), in the rat dorsal root ganglia was examined by double in situ hybridization technique. About 90% and 30% of PPTA mRNA-positive neurons expressed mu- and kappa-OPR mRNAs at high level, respectively. However, only about 3% of PPTA mRNA-positive neurons expressed delta-OPR mRNA at high level. These results suggest that mu- and kappa-OPRs exist on most of and a part of the primary afferent terminals containing SP, respectively. On the other hand, among the neurons which highly expressed mu-, delta- or kappa-OPR mRNA, PPTA mRNA was not expressed in about 58%, 95% or 24% of those neurons, respectively. These findings suggest the possibility that OPRs co-exist with other neurotransmitters and/or neuromodulators than SP in the primary afferent neurons.

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.

In situ hybridization study of μ- and κ-opioid receptor mRNAs in the rat spinal cord and dorsal root ganglia

Abstract Distributions of μ- and κ-opioid receptor mRNAs in the lumbar spinal cord and dorsal root ganglia of the adult rat were examined using the in situ hybridization technique. In the lumbar spinal cord, μ-opioid receptor mRNA was expressed intensely in laminae I, II and VIII. On the other hand, κ-opioid receptor mRNA was expressed intensely in laminae I and II, and moderately throughout laminae III-VIII. In the dorsal root ganglia, μ-opioid receptor mRNA was intensely expressed and κ-opioid receptor mRNA was expressed in a smaller number of cells than μ-opioid receptor mRNA.

Expression of μ- and κ-, but not δ-, opioid receptor mRNAs is enhanced in the spinal dorsal horn of the arthritic rats

&NA; The expression of the mRNAs for &mgr;‐, &dgr;‐ and &kgr;‐opioid receptors was studied in the lumbar spinal cord of the rats with the inflammation at their unilateral hindpaw using in situ hybridization technique. On 11 days after the first adjuvant inoculation, &mgr;‐ and &kgr;‐opioid receptor mRNA levels in laminae I–II of the spinal dorsal horn ipsilateral to the inflamed hindpaw were increased to 135.3 ± 6.4% and 130.3 ± 5.7%, respectively, compared with the contralateral side. At this time point, no significant differences in the &mgr;‐ and &kgr;‐opioid receptor mRNA expression were observed between ipsi‐ and contralateral sides of other laminae. On the other hand, no significant change was observed in the &dgr;‐opioid receptor mRNA expression throughout the laminae I–IX at any time points examined. These findings suggest the increase in the synthesis of &mgr;‐ and &kgr;‐, but not &dgr;‐, opioid receptors in the spinal laminae I–II during sustained inflammatory pain.

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.

Expression of aldose reductase and sorbitol dehydrogenase genes in Schwann cells isolated from rat : effects of high glucose and osmotic stress

To investigate the polyol pathway activity in Schwann cells, we determined the mRNA levels of aldose reductase (AR) and sorbitol dehydrogenase (SDH) in cultured cells under hyperglycemic or hyperosmotic conditions using competitive RT-PCR technique. The expressions of AR and SDH mRNAs in Schwann cells were unaltered by high (30 mM) glucose content in the medium. On the other hand, osmotic stress elicited significant increases in AR mRNA without any effect on SDH mRNA expression. The levels of AR mRNA determined by this RT-PCR system were significantly correlated with AR activity, as well as the levels of sorbitol accumulated in Schwann cells cultured under hyperosmotic conditions. These findings suggest that in contrast to the induction of AR expression by osmotic stress, high glucose per se does not up-regulate expression of the enzymes constituting the polyol pathway in Schwann cells. The RT-PCR system developed in this study may be a useful tool in ascertaining the relative contributions of AR and SDH to the metabolic derangements leading to diabetic complications.

Clinical analysis of aldose reductase for differential diagnosis of the pathogenesis of diabetic complication

A two-site immunoassay system for evaluating tissue levels of aldose reductase was developed using monoclonal and polyclonal antibodies to the recombinant human enzyme expressed in a baculovirus system. The calibration graph of this system was linear for concentrations of aldose reductase ranging from 0 to 16 ng ml -1 . The detection limit was 0.034 ng ml -1 . The coefficients of variation within and between assays were 3.7% and 4.8%, respectively. Analytical recovery was 101-106%. The amount of aldose reductase expressed in human tissues was measured by this system. Higher levels of aldose reductase were found in kidney medulla, sciatic nerve and lens indicating that the enzyme level was higher in the target organs of diabetic complications. The aldose reductase level of sciatic nerve was well correlated with that of erythrocytes (r=0.62. p<0.05). When erythrocyte aldose reductase was determined in 160 patients with a diabetic duration for less than 10 years, the enzyme level in patients with neuropathy was significantly higher than in those without any complications. The incidence of neuropathy in these patients was significantly increased in proportion to the enzyme levels (p<0.05). In this study, it has become apparent that the aldose reductase level in erythrocytes of patients with short diabetic duration (<10 years) is associated with the presence of neuropathy. It was also suggested that the immunoassay system will provide useful clinical information to optimize administration of aldose reductase inhibitors for effective prevention and treatment of diabetic complications.

Kainic acid induces leukemia inhibitory factor mRNA expression in the rat brain: differences in the time course of mRNA expression between the dentate gyrus and hippocampal CA1/CA3 subfields.

Leukemia inhibitory factor (LIF) is a pluripotent cytokine which affects the survival and differentiation of various types of cells both in the hematopoietic and nervous systems. In this study, the time course and localization of LIF mRNA expression following kainic acid-induced seizures were examined by northern blot analyses and in situ hybridization. Northern blot analyses demonstrated that intraperitoneal injection of kainic acid at a convulsive dose induced LIF mRNA expression intensely in the hippocampus and moderately to weakly in the cerebral cortex, thalamus and hypothalamus. The expression peaked at 8-24 h after the injection in the hippocampus and cerebral cortex and at 8 h in the thalamus and hypothalamus. In situ hybridization revealed different time courses of LIF mRNA expression depending on the area of the hippocampus; that is, the expression peaked at 10 h in the granule cell layer of the dentate gyrus, then at 12 h in the polymorph and molecular layers of the dentate gyrus, and finally at 12-24 h in the strata oriens and radiatum of the CA1 and CA3 subfields. It is worth noting that the expression of LIF mRNA was intense in the dentate gyrus, the region where neurogenesis and aberrant network reorganization have been shown to be induced by seizures. The upregulation of LIF mRNA expression in the dentate granule cell layer followed by that in the dentate polymorph and molecular layers may be involved in activity-dependent neurogenesis in the granule cell layer and ectopic migration of granule cells to the polymorph and molecular layers in the dentate gyrus.

Analysis of gene expression of aldose reductase and sorbitol dehydrogenase in rat Schwann cells by competitive RT-PCR method using non-homologous DNA standards

Aldose reductase (AR) and sorbitol dehydrogenase (SDH) are the enzymes constituting the polyol pathway, an alternate route of glucose metabolism. A wealth of experimental data has indicated the involvement of the polyol pathway in the pathogenesis of diabetic complications. However, there has been surprisingly little research on the relative abundance of SDH to AR in the tissues affected in diabetes. We therefore developed a competitive RT-PCR system to simultaneously determine the mRNA levels of these two enzymes in small amounts of samples, and studied their expression in Schwann cells isolated from adult rat sciatic nerves. Although both AR and SDH mRNA were expressed in the Schwann cells, the levels of SDH cDNA were much lower than those of AR cDNA. The induction of AR mRNA expression in the Schwann cells under hyperosmotic conditions was similarly detected by Northern blot analysis and our competitive RT-PCR method. The RT-PCR system developed in this study may be a useful tool in ascertaining the relative contributions of AR and SDH to the metabolic derangements resulting from the acceleration of polyol pathway activity in the target organ of diabetic complications.