Postsynaptic modulation of AMPA receptor-mediated synaptic responses and LTP by the type 3 ryanodine receptor.

The precise function of ryanodine receptors (RyRs) in synaptic transmission is unknown, but three of their subtypes are expressed in the brain. We examined the roleof RyRs in excitatory synaptic transmission in hippocampal slices, using type 3 RyR (RyR3)-deficient mice. The alpha-amino-3-hydroxy-5-methyl-4-isoxozolepropionic acid (AMPA) receptor-mediated basal synaptic responses in the CA1 region of mutant mice were smaller than those of wild-type mice, while there was no difference in N-methyl-d-aspartate receptor-mediated responses, suggesting selective postsynaptic modification of AMPA receptors by RyR3. The expression of synaptic AMPA receptor subunits examined by Western blotting or immunohistochemistry was indistinguishable, suggesting that the smaller AMPA synaptic responses in mutant mice were not due to the reduced number of synaptic AMPA receptors. Although the initial potentiation following tetanic stimulation of afferent fibers was similar, long-term potentiation (LTP) was smaller in mutant mice. There were no differences in presynaptic electrophysiological properties. We thus conclude that RyR3 postsynaptically regulates the properties of AMPA receptors and LTP.

Fas ligand-mediated exocrinopathy resembling Sjögren's syndrome in mice transgenic for IL-10.

Although IL-10 has been implicated in the pathogenesis of several autoimmune diseases, the mechanisms by which this cytokine mediates inflammatory lesions remain to be elucidated. Exocrine gland destruction is an important early step in the development of Sjögren's syndrome. To better understand the role of IL-10 in Sjögren's syndrome, we made transgenic mice in which the mouse IL-10 gene was regulated by the human salivary amylase promoter. Transgenic expression of IL-10 induced apoptosis of glandular tissue destruction and lymphocyte infiltration consisting primarily of Fas-ligand (FasL)+ CD4+ T cells, as well as in vitro up-regulation of FasL expression on T cells. These data suggest that overexpression of IL-10 in the glands and their subsequent Fas/FasL-mediated bystander tissue destruction is a causal factor in the development of this disease.

Structure and expression of mitsugumin29 gene.

Recently mitsugumin29 unique to the triad junction in skeletal muscle was identified as a novel member of the synaptophysin family; the members of this family have four transmembrane segments and are distributed on intracellular vesicles. In this study, we isolated and analyzed mouse mitsugumin29 cDNA and genomic DNA containing the gene. The mitsugumin29 gene mapped to the mouse chromosome 3 F3-H2 is closely related to the synaptophysin gene in exon-intron organization, which indicates their intimate relationship in molecular evolution. RNA blot hybridization and immunoblot analysis revealed that mitsugumin29 is expressed abundantly in skeletal muscle and at lower levels in the kidney. Immunofluorescence microscopy demonstrated that mitsugumin29 exists specifically in cytoplasmic regions of the proximal and distal tubule cells in the kidney. The results obtained may suggest that mitsugumin29 is involved in the formation of specialized endoplasmic reticulum systems in skeletal muscle and renal tubule cells.

Mitsugumin29, a novel synaptophysin family member from the triad junction in skeletal muscle.

In skeletal muscle, excitation-contraction (E-C) coupling requires the conversion of the depolarization signal of the invaginated surface membrane, namely the transverse (T-) tubule, to Ca2+ release from the sarcoplasmic reticulum (SR). Signal transduction occurs at the junctional complex between the T-tubule and SR, designated as the triad junction, which contains two components essential for E-C coupling, namely the dihydropyridine receptor as the T-tubular voltage sensor and the ryanodine receptor as the SR Ca2+-release channel. However, functional expression of the two receptors seemed to constitute neither the signal-transduction system nor the junction between the surface and intracellular membranes in cultured cells, suggesting that some as-yet-unidentified molecules participate in both the machinery. In addition, the molecular basis of the formation of the triad junction is totally unknown. It is therefore important to examine the components localized to the triad junction. Here we report the identification using monoclonal antibody and primary structure by cDNA cloning of mitsugumin29, a novel transmembrane protein from the triad junction in skeletal muscle. This protein is homologous in amino acid sequence and shares characteristic structural features with the members of the synaptophysin family. The subcellular distribution and protein structure suggest that mitsugumin29 is involved in communication between the T-tubular and junctional SR membranes.

A region of the ryanodine receptor critical for excitation-contraction coupling in skeletal muscle.

Ca2+ release mediated by the ryanodine receptor (RyR) regulates many important cell functions including excitation-contraction (E-C) coupling in skeletal muscle, by which membrane depolarization controls the opening of RyR via the dihydropyridine receptor. Among the three RyR subtypes, RyR-1 mediates skeletal muscle E-C coupling, whereas RyR-2 and RyR-3 cannot substitute for RyR-1. We carried out expression experiments using cultured mutant skeletal myocytes not having intrinsic intracellular Ca2+ release channels to study the structure-function relationship of amino acid residues 1303-1406 in RyR-1 (D2 region). In this region the amino acid sequences are highly divergent between RyR-1 and RyR-2, and the corresponding sequence is lacking in RyR-3. Expression of RyR-1 but not of RyR-2 rescued E-C coupling in the mutant cells. Deletion of either the entire D2 region or its N-terminal half from RyR-1 preserved the function of RyR-1 as a Ca2+ release channel but resulted in the loss of E-C coupling. Substitution of the D2 region for the corresponding sequence of RyR-2 had no effect on the function of RyR-1. These results indicate that the presence of the D2 region is critical for E-C coupling in skeletal muscle, although the D2 region alone cannot determine the functional difference between RyR-1 and RyR-2.

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.

Increased expression of human thioredoxin/adult T cell leukemia-derived factor in Sjögren's syndrome.

OBJECTIVE: To determine the involvement of human thioredoxin/adult T cell leukemia-derived factor TRX/ADF) in Sjögren's syndrome (SS) and the correlation with Epstein-Barr virus (EBV). METHODS: Indirect immunohistochemical techniques and reverse transcriptase polymerase chain reaction were utilized to analyze TRX/ADF expression and the presence of EBV, using 6 normal tissues and 23 surgical specimens. The kinetics of expression of TRX/ADF induced by EBV was examined in vitro with peripheral blood B cells from EBV-seronegative donors. RESULTS: Marked expression of TRX/ADF was found in the infiltrating B cells and the epithelial cells of salivary gland tissues from patients with SS (11 of 12 cases), but not in those from patients with other salivary gland inflammatory conditions (0 of 11 cases) or those of normal individuals (0 of 6 cases). In immunohistologic analyses, a striking topographic correlation between TRX/ADF and EBV was found. The coexistence of TRX/ADF messenger RNA and EBV DNA was detected by polymerase chain reaction (r = 0.75, P < 0.01). Peripheral blood B cells from EBV-seronegative donors showed de novo synthesis of TRX/ADF following in vitro infection with EBV. EBV-infected B cell lines all expressed TRX/ADF. TRX/ADF was not detected in non-EBV-infected cells. Tumors in SCID mice reconstituted with mononuclear cells of salivary glands from SS patients, which were composed of human B cells carrying EBV DNA, were positive for TRX/ADF. CONCLUSION: These findings suggest that TRX/ADF expression closely reflects the intracellular event of EBV reactivation in SS. This is also the first report to show the ectopic in vivo expression of TRX/ADF in human autoimmune disease.

Expression of cell adhesion molecules in the salivary and lacrimal glands of Sjogren's syndrome.

We attempted to determine whether cell adhesion molecules, including vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin (endothelial-leukocyte adhesion molecule-1; ELAM-1), are involved in the lymphoid cell infiltration of the salivary and lacrimal glands in Sjogren's syndrome (SS) patients. Both immunohistochemical analysis and the reverse-transcripts polymerase chain reaction (RT-PCR) were used to analyze the expression of VCAM-1, ICAM-1, ELAM-1, very late antigen 4 (VLA-4 [alpha 4,beta 1]), lymphocyte function-associated antigen-1 (LFA-1), interferon-gamma (IFN-gamma), tumor necrosis factor (TNF), and interleukin-1 beta (IL-1 beta). Immunohistochemical analysis of salivary gland biopsies from SS patients showed a marked expression of VCAM-1 and ICAM-1 in the venules surrounded by infiltrated CD4+ CD45RO+ T cells. E-selectin was expressed on vascular endothelium with weak intensity. Increased levels of VCAM-1, ICAM-1, IFN-gamma, and IL-1 beta mRNA were demonstrated by RT-PCR, whereas E-selectin mRNA were weakly expressed in SS lacrimal and salivary gland tissues. This is in contrast with strong expression of ELAM-1 in IL-1 beta-stimulated human umbilical vascular endothelial cells (HUVEC) in vitro. Cytokine-mediated up-regulation of VCAM-1 and ICAM-1 that facilitates the recruitment of VLA-4 and LFA-1 expressing T cells might contribute to lymphoid cell infiltration in the salivary and lacrimal glands in SS.