Characterization of ryanodine receptor and Ca2+-ATPase isoforms in the thermogenic heater organ of blue marlin (Makaira nigricans)

Abstract

SUMMARY A thermogenic organ is found beneath the brain of billfishes(Istiophoridae), swordfish (Xiphiidae) and the butterfly mackerel(Scombridae). The heater organ has been shown to warm the brain and eyes up to 14°C above ambient water temperature. Heater cells are derived from extraocular muscle fibers and express a modified muscle phenotype with an extensive transverse-tubule (T-tubule) network and sarcoplasmic reticulum (SR)enriched in Ca2+-ATPase (SERCA) pumps and ryanodine receptors(RyRs). Heater cells have a high mitochondria content but have lost most of the contractile myofilaments. Thermogenesis has been hypothesized to be associated with release and reuptake of Ca2+. In this study,Ca2+ fluxes in heater SR vesicles derived from blue marlin(Makaira nigricans) were measured using fura-2 fluorescence. Upon the addition of MgATP, heater SR vesicles rapidly sequestered Ca2+. Uptake of Ca2+ was thapsigargin sensitive, and maximum loading ranged between 0.8 μmol Ca2+ mg-1 protein and 1.0μmol Ca2+ mg-1 protein. Upon the addition of 10 mmol l-1 caffeine or 350 μmol l-1 ryanodine, heater SR vesicles released only a small fraction of the loaded Ca2+. However, ryanodine could elicit a much larger Ca2+ release event when the activity of the SERCA pumps was reduced. RNase protection assays revealed that heater tissue expresses an RyR isoform that is also expressed in fish slow-twitch skeletal muscle but is distinct from the RyR expressed in fish fast-twitch skeletal muscle. The heater and slow-twitch muscle RyR isoform has unique physiological properties. In the presence of adenine nucleotides, this RyR remains open even though cytoplasmic Ca2+ is elevated, a condition that normally closes RyRs. The fast Ca2+sequestration by the heater SR, coupled with a physiologically unique RyR, is hypothesized to promote Ca2+ cycling, ATP turnover and heat generation. A branch of the oculomotor nerve innervates heater organs, and, in this paper, we demonstrate that heater cells contain large `endplate-like'clusters of acetylcholine receptors that appear to provide a mechanism for nervous control of thermogenesis.