Biology subjects

Nakada, T.

Publications and source records attributed to Nakada, T..

3 recordsLinked to original sources

bioRxiv2026

Zebrafish larval nitrogen excretion is flexible and resilient to loss of rhesus glycoproteins

Nitrogenous waste excretion is essential for all developmental stages of fish. Embryonic fish excrete urea, transitioning to cutaneous and later branchial ammonia excretion. In zebrafish, ammonia excretion involves rhesus glycoproteins Rhbg and Rhcgb in keratinocytes and ionocytes, but the developmental moment they appear in the gill remains unclear. Potential redundancy between Rhbg and Rhcgb in ammonia excretion is also not fully investigated, nor is the difference in response to low pH. We hypothesized that rhesus glycoproteins are partially redundant, and that they differ in their response to low pH as ammonia excretion enables ionocytes to exchange Na+ and H+ (Rh-NHE-metabolon). We predicted that a loss of rhbg or rhcgb induces compensatory responses. We characterized the transition from urea to branchial ammonia excretion from 0 to 8 days-post fertilization (dpf) and the response to pH 5.0 on the expression and localization of rhesus glycoproteins in control zebrafish and rhbg or rhcgb-crispants. Effects of high external ammonia (HEA, 500 M NH4Cl) and 10 mM HEPES-buffering were further characterized in rhcgb-crispants. Rhag and Rhbg appeared in the gill at 5 dpf, while Rhcgb appeared at 6 dpf. A loss of rhbg or rhcgb did not impact baseline N-excretion, illustrating that zebrafish can maintain ammonia excretion without the full complement of rhesus glycoproteins. We observed no compensatory increase in rhesus glycoproteins, but expression of the transporter hippocampus-abundant transcript 1b increased. HEA-exposed rhcgb-crispants switched to urea as primary nitrogen waste. Together, these findings underline the plasticity of the larval in dealing with nitrogenous waste.

physiology↗

bioRxiv2022

High-throughput platform for depolarization-induced Ca2+ release for skeletal muscle disease mutation validation and drug discovery

In skeletal muscle excitation-contraction (E-C) coupling, depolarization of the plasma membrane triggers Ca2+ release from the sarcoplasmic reticulum (SR), referred to as depolarization-induced Ca2+ release (DICR). DICR occurs via the type 1 ryanodine receptor (RyR1), which physically interacts with the dihydropyridine receptor Cav1.1 subunit in specific machinery formed with additional essential components including {beta}1a, Stac3 adaptor protein and junctophilins. Exome sequencing has accelerated the discovery of many novel mutations in genes encoding DICR machinery in various skeletal muscle diseases. However, functional validation is time-consuming because it must be performed in a skeletal muscle environment. In this study, we established a platform of the reconstituted DICR in HEK293 cells. The essential components were effectively transduced into HEK293 cells expressing RyR1 using baculovirus vectors, and Ca2+ release was quantitatively measured with R-CEPIA1er, a fluorescent ER Ca2+ indicator, without contaminant of extracellular Ca2+ influx. In these cells, [K+]-dependent Ca2+ release was triggered by chemical depolarization with the aid of inward rectifying potassium channel, indicating a successful reconstitution of DICR. Using the platform, we evaluated several Cav1.1 mutations that are implicated in malignant hyperthermia and myopathy. We also tested several RyR1 inhibitors; whereas dantrolene and Cpd1 inhibited DICR, procaine had no effect. Furthermore, twitch potentiators such as perchlorate and thiocyanate shifted the voltage dependence of DICR to more negative potentials without affecting Ca2+-induced Ca2+ release. These results well reproduced the findings with the muscle fibers and the cultured myotubes. Since the procedure is simple and reproducible, the reconstituted DICR platform will be highly useful for validation of mutations and drug discovery for skeletal muscle diseases. SummaryMutations in essential components for depolarization-induced Ca2+ release (DICR) are implicated into various skeletal muscle diseases. Murayama et al. establish a reconstituted DICR platform in nonmuscle cells for evaluation of disease-causing mutations and drug discovery.

physiology↗

bioRxiv2018

Aquaporin-4 facilitator TGN-073 demonstrates novel analgesic activity

During pre-clinical development, we tested the novel, internally developed AQP-4 facilitator TGN-073 for its effect in a rodent pain model. Therein, TGN-073 was found to exert a strong analgesic effect. Following a single 200 mg/kg (i.p.) administration of TGN-073, a virtually complete block in the acetic acid writhing test was observed. Subsequent in vitro tests demonstrated that TGN-073 had no binding affinity for the -opioid or NK-1 receptors. Accordingly, we suspect TGN-073 or other AQP-4 facilitators may be developed into potent non-opioid analgesic agents. Given the potential significance of this discovery, we feel it should be openly shared with the scientific community.

pharmacology and toxicology↗