Miyuki Nishi

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.

TRPM2-mediated Ca2+ influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration

Reactive oxygen species (ROS) induce chemokines responsible for the recruitment of inflammatory cells to sites of injury or infection. Here we show that the plasma membrane Ca2+-permeable channel TRPM2 controls ROS-induced chemokine production in monocytes. In human U937 monocytes, hydrogen peroxide (H2O2) evokes Ca2+ influx through TRPM2 to activate Ca2+-dependent tyrosine kinase Pyk2 and amplify Erk signaling via Ras GTPase. This elicits nuclear translocation of nuclear factor-κB essential for the production of the chemokine interleukin-8 (CXCL8). In monocytes from Trpm2-deficient mice, H2O2-induced Ca2+ influx and production of the macrophage inflammatory protein-2 (CXCL2), the mouse CXCL8 functional homolog, were impaired. In the dextran sulfate sodium-induced colitis inflammation model, CXCL2 expression, neutrophil infiltration and ulceration were attenuated by Trpm2 disruption. Thus, TRPM2 Ca2+ influx controls the ROS-induced signaling cascade responsible for chemokine production, which aggravates inflammation. We propose functional inhibition of TRPM2 channels as a new therapeutic strategy for treating inflammatory diseases.

Facilitation of long-term potentiation and memory in mice lacking nociceptin receptors

The peptide nociceptin (also named orphanin FQ) acts in the brain to produce various pharmacological effects, including hyperalgesia and hypolocomotion,. The nociceptin receptor uses guanine-nucleotide-binding proteins to mediate the inhibition of adenylyl cyclase, the activation of potassium channels and inhibition of calcium channels. It has been shown using knockout mice that the nociceptin receptor is not required for regulation of nociceptive responses or locomotion activity, but modulates the auditory function. Here we show that mice lacking the nociceptin receptor possess greater learning ability and have better memory than control mice. Histological analysis revealed the expression of both the nociceptin precursor and the nociceptin receptor in the hippocampus, thought to take part in aspects of learning and memory. Moreover, the receptor-deficient mice showed larger long-term potentiation in the hippocampal CA1 region than control mice, without apparent changes in presynaptic or postsynaptic electrophysiological properties. These results show that the loss of the nociceptin receptor results in a gain-of-function mutation in both the memory process and the long-term potentiation mechanism in CA1, perhaps as a result of altered intracellular signal transduction systems in neurons.

Unrestrained nociceptive response and disregulation of hearing ability in mice lacking the nociceptin/orphaninFQ receptor.

In the G‐protein‐coupled receptor superfamily, the opioid receptor subfamily is constituted of the three distinct opioid receptors (namely δ‐, μ‐ and κ‐subtypes) and the receptor for nociceptin (also designated orphaninFQ). The members of the opioid receptor subfamily were known to mediate a variety of cellular inhibitory effects. The three opioid receptors are known to play central roles in mediating analgesia and many other physiological activities; however, the nociceptin receptor was identified recently and less is known about its physiological roles. Here we report the generation and characterization of mice lacking the nociceptin receptor. The knockout mice showed no significant differences in nociceptive threshold and locomotor activity compared with control mice, but they lost nociceptin‐induced behavioral responses. These results indicate that the nociceptin system is not essential for regulation of nociception or locomotor activity. On the other hand, we found insufficient recovery of hearing ability from the adaptation to sound exposure in the mutant mice. Thus, the nociceptin system appears to participate in the regulation of the auditory 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.

EMBRYONIC LETHALITY AND ABNORMAL CARDIAC MYOCYTES IN MICE LACKING RYANODINE RECEPTOR TYPE 2

The ryanodine receptor type 2 (RyR‐2) functions as a Ca2+‐induced Ca2+ release (CICR) channel on intracellular Ca2+ stores and is distributed in most excitable cells with the exception of skeletal muscle cells. RyR‐2 is abundantly expressed in cardiac muscle cells and is thought to mediate Ca2+ release triggered by Ca2+ influx through the voltage‐gated Ca2+ channel to constitute the cardiac type of excitation–contraction (E–C) coupling. Here we report on mutant mice lacking RyR‐2. The mutant mice died at approximately embryonic day (E) 10 with morphological abnormalities in the heart tube. Prior to embryonic death, large vacuolate sarcoplasmic reticulum (SR) and structurally abnormal mitochondria began to develop in the mutant cardiac myocytes, and the vacuolate SR appeared to contain high concentrations of Ca2+. Fluorometric Ca2+ measurements showed that a Ca2+ transient evoked by caffeine, an activator of RyRs, was abolished in the mutant cardiac myocytes. However, both mutant and control hearts showed spontaneous rhythmic contractions at E9.5. Moreover, treatment with ryanodine, which locks RyR channels in their open state, did not exert a major effect on spontaneous Ca2+ transients in control cardiac myocytes at E9.5–11.5. These results suggest no essential contribution of the RyR‐2 to E–C coupling in cardiac myocytes during early embryonic stages. Our results from the mutant mice indicate that the major role of RyR‐2 is not in E–C coupling as the CICR channel in embryonic cardiac myocytes but it is absolutely required for cellular Ca2+ homeostasis most probably as a major Ca2+ leak channel to maintain the developing SR.

Membrane repair defects in muscular dystrophy are linked to altered interaction between MG53, caveolin-3, and dysferlin.

Defective membrane repair can contribute to the progression of muscular dystrophy. Although mutations in caveolin-3 (Cav3) and dysferlin are linked to muscular dystrophy in human patients, the molecular mechanism underlying the functional interplay between Cav3 and dysferlin in membrane repair of muscle physiology and disease has not been fully resolved. We recently discovered that mitsugumin 53 (MG53), a muscle-specific TRIM (Tri-partite motif) family protein (TRIM72), contributes to intracellular vesicle trafficking and is an essential component of the membrane repair machinery in striated muscle. Here we show that MG53 interacts with dysferlin and Cav3 to regulate membrane repair in skeletal muscle. MG53 mediates active trafficking of intracellular vesicles to the sarcolemma and is required for movement of dysferlin to sites of cell injury during repair patch formation. Mutations in Cav3 (P104L, R26Q) that cause retention of Cav3 in Golgi apparatus result in aberrant localization of MG53 and dysferlin in a dominant-negative fashion, leading to defective membrane repair. Our data reveal that a molecular complex formed by MG53, dysferlin, and Cav3 is essential for repair of muscle membrane damage and also provide a therapeutic target for treatment of muscular and cardiovascular diseases that are linked to compromised membrane repair.

Generation and Characterization of Mutant Mice Lacking Ryanodine Receptor Type 3

The ryanodine receptor type 3 (RyR-3) functions as a Ca2+-induced Ca2+ release (CICR) channel and is distributed in a wide variety of cell types including skeletal muscle and smooth muscle cells, neurons, and certain non-excitable cells. However, the physiological roles of RyR-3 are totally unclear. To gain an insight into the function of RyR-3 in vivo, we have generated mice lacking RyR-3 by means of the gene targeting technique. The mutant mice thus obtained showed apparently normal growth and reproduction. Although Ca2+-induced Ca2+ release from intracellular Ca2+ stores of the mutant skeletal muscle differed in Ca2+ sensitivity from that of wild-type muscle, excitation-contraction coupling of the mutant muscle seemed to be normal. Moreover, we could not find any significant disturbance in the smooth muscle and lymphocytes from the mutant mice. On the other hand, the mutant mice showed increased locomotor activity, which was about 2-fold greater than that of the control mice. These results indicate that the loss of RyR-3 causes no gross abnormalities and suggest that the lack of RyR-3-mediated Ca2+ signaling results in abnormalities of certain neurons in the central nervous system.

Deficiency of triad junction and contraction in mutant skeletal muscle lacking junctophilin type 1

In skeletal muscle excitation–contraction (E–C) coupling, the depolarization signal is converted from the intracellular Ca2+ store into Ca2+ release by functional coupling between the cell surface voltage sensor and the Ca2+ release channel on the sarcoplasmic reticulum (SR). The signal conversion occurs in the junctional membrane complex known as the triad junction, where the invaginated plasma membrane called the transverse-tubule (T-tubule) is pinched from both sides by SR membranes. Previous studies have suggested that junctophilins (JPs) contribute to the formation of the junctional membrane complexes by spanning the intracellular store membrane and interacting with the plasma membrane (PM) in excitable cells. Of the three JP subtypes, both type 1 (JP-1) and type 2 (JP-2) are abundantly expressed in skeletal muscle. To examine the physiological role of JP-1 in skeletal muscle, we generated mutant mice lacking JP-1. The JP-1 knockout mice showed no milk suckling and died shortly after birth. Ultrastructural analysis demonstrated that triad junctions were reduced in number, and that the SR was often structurally abnormal in the skeletal muscles of the mutant mice. The mutant muscle developed less contractile force (evoked by low-frequency electrical stimuli) and showed abnormal sensitivities to extracellular Ca2+. Our results indicate that JP-1 contributes to the construction of triad junctions and that it is essential for the efficiency of signal conversion during E–C coupling in skeletal muscle.

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.