Hiroshi Takeshima

cDNA cloning and regional distribution of a novel member of the opioid receptor family

We have cloned a cDNA for a novel member of the opioid receptor family, designated as ROR‐C, from the rat cerebrum cDNA library using the probe derived from the δ‐opioid receptor subtype cDNA. The deduced amino acid sequence of ROR‐C shows high homology with those of ROR‐A (rat δ‐opioid receptor subtype), ROR‐B (rat μ‐subtype) and ROR‐D (rat k‐subtype). RNA blot hybridization and in situ hybridization analysis revealed that ROR‐C mRNA is expressed in discrete regions of the rat central nervous system.

Primary structures and expression from cDNAs of rat opioid receptor δ-and μ-subtypes

The complete amino acid sequences of rat opioid receptors (designated as ROR‐A and ROR‐B) have been deduced by cloning and sequencing the cDNAs. The ligand‐binding properties of ROR‐A and ROR‐B expressed from the cloned cDNAs in Chinese hamster ovary cells correspond most closely to those of the pharmacologically defined δ‐ and μ‐opioid receptor subtypes, respectively. RNA blot hybridization analysis revealed that cerebrum and brainstem contain both ROR‐A and ROR‐B mRNAs, whereas neither ROR‐A nor ROR‐B mRNAs can be detected in cerebellum.

Isolation and characterization of a gene for a ryanodine receptor/calcium release channel in Drosophila melanogaster

The nucleotide sequence of a 25.7 kilobase Drosophila melanogaster genomic DNA segment containing a gene for a ryanodine receptor/calcium release channel homologue has been determined. Computer analysis and partial cDNA cloning revealed 26 exons comprising the protein‐coding sequence in this gene. The predicted protein is homologous in amino acid sequence and shares characteristic structural features with the mammalian ryanodine receptors. In blot hybridization analysis, a ~16 kilobase RNA species was identified abundantly in a 6–12 h embryo as the transcript from this gene. In situ hybridization to polytene chromosomes indicated that this gene locates at band position 44F on the second chromosome.

cDNA cloning and pharmacological characterization of an opioid receptor with high affinities for κ‐subtype‐selective ligands

The amino acid sequence of a rat opioid receptor, designated as ROR‐D, has been deduced by cloning and sequencing the cDNA. The ROR‐D expressed from the cDNA exhibits high affinities for ligands selective for the opioid receptor κ‐subtype and low affinities for ligands selective for the δ‐ and μ‐subtypes. RNA blot hybridization analysis indicated that ROR‐D mRNA is distributed in cerebrum and brainstem but not in cerebellum.

CO-EXPRESSION IN CHO CELLS OF TWO MUSCLE PROTEINS INVOLVED IN EXCITATION-CONTRACTION COUPLING

SummaryRyanodine receptors and dihydropyridine receptors are located opposite each other at the junctions between sarcoplasmic reticulum and either the surface membrane or the transverse tubules in skeletal muscle. Ryanodine receptors are the calcium release channels of the sarcoplasmic reticulum and their cytoplasmic domains form the feet, connecting sarcoplasmic reticulum to transverse tubules. Dihydropyridine receptors are L-type calcium channels that act as the voltage sensors of excitation-contraction coupling: they sense surface membrane and tranverse tubule depolarization and induce opening of the sarcoplasmic reticulum release channels. In skeletal muscle, ryanodine receptors are arranged in extensive arrays and dihydropyridine receptors are grouped into tetrads, which in turn are associated with the four subunits of ryanodine receptors. The disposition allows for a direct interaction between the two sets of molecules.CHO cells were stably transformed with plasmids for skeletal muscle ryanodine receptors and either the skeletal dihydropyridine receptor, or a skeletal-cardiac dihydropyridine receptor chimera (CSk3) which can functionally substitute for the skeletal dihydropyridine receptor, in addition to plasmids for the α2, β and γ subunits. RNA blot hybridization gave positive results for all components. Immunoblots, ryanodine binding, electron microscopy and exposure to caffeine show that the expressed ryanodine receptors forms functional tetrameric channels, which are correctly inserted into the endoplasmic reticulum membrane, and form extensive arrays with the same spacings as in skeletal muscle. Since formation of arrays does not require coexpression of dihydropyridine receptors, we conclude that self-aggregation is an independent property of ryanodine receptors. All dihydropyridine receptor-expressing clones show high affinity binding for dihydropyridine and immunolabelling with antibodies against dihydropyridine receptor. The presence of calcium currents with fast kinetics and immunolabelling for dihydropyridine receptors in the surface membrane of CSk3 clones indicate that CSk3-dihydropyridine receptors are appropriately targeted to the cells plasmalemma. The expressed skeletal-type dihydropyridine receptors, however, remain mostly located within perinuclear membranes. In cells coexpressing functional dihydropyridine receptors and ryanodine receptors, no junctions between feet-bearing endoplasmic reticulum elements and surface membrane are formed, and dihydropyridine receptors do not assemble into tetrads. A separation between dihydropyridine receptors and ryanodine receptors is not unique to CHO cells, but is found also in cardiac muscle, in muscles of invertebrates and, under certain conditions, in skeletal muscle. We suggest that failure to form junctions in co-transfected CHO cell may be due to lack of an essential protein necessary either for the initial docking of the endoplasmic reticulum to the surface membrane or for maintaining the interaction between dihydropyridine receptors and ryanodine receptors. We also conclude that formation of tetrads requires a close interaction between dihydropyridine receptors and ryanodine receptors.

Opioid μ- and κ-receptor mediate phospholipase C activation through Gi1 in Xenopus oocytes

Abstract In the Xenopus oocytes expressing μ or κ-opioid receptors, agonist-induced currents were observed only when the oocyte was coinjected with Gi1 α RNA and pretreated with K-252a, a potent inhibitor of protein kinases. The evoked currents were abolished by i intracellular injection of EGTA or inositol 1,4,5-trisphosphate and the current-voltage relationship revealed that they are mediated through typical calcium-dependent chloride channels. These findings suggest that the μ- and κ-receptors mediate phospholipase C activation through Gi1 α, and that these receptor mechanisms including downstream signalings might be inhibited by phosphorylation in vivo in the Xenopus oocyte.

μ Opioid receptor: Expression and vagotomy-induced depletion of the mRNA in medullary preganglionic neurons

By in situ hybridization with digoxigenin-labeled RNA probes, distinct expression of mu opioid receptor mRNA was found in rat medullary preganglionic neurons for vagal parasympathetic outflow. Two days or more following unilateral vagotomy, the expression was almost completely abolished on the operated side. The results, taken together with previous findings indicating proximodistal axonal transport of radioligand-labeled opioid receptors, suggest that the medullary preganglionic neurons thus identified are the source for mu opioid receptor acting in cardiorespiratory and gastrointestinal tissues.