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

Jeffery M. Morrissette^*, Jens P. G. Franck and Barbara A. Block

Stanford University, Hopkins Marine Station, Pacific Grove, CA 93950, USA
Present address: Occidental College, Los Angeles, CA 90041, USA

View larger version (36K): [in a new window]   Fig. 1. RNase protection assay reveals the expression of RyR1-slow in heater tissue. A 32P-labeled RNA probe, synthesized from a blue marlin extraocular muscle clone, hybridized to slow-twitch skeletal muscle and heater organ but not to fast-twitch skeletal muscle. This isoform was named RyR1-slow to distinguish it from RyR1-fast, which is expressed in fish fast-twitch skeletal muscle. The probe was hybridized to 20 µg total RNA from each tissue. Yeast, control RNA; TFSB, toadfish fast-twitch swimbladder muscle; BMCard, blue marlin cardiac muscle; SFHO, swordfish heater organ; BMST, blue marlin slow-twitch skeletal muscle; BMFT, blue marlin fast-twitch skeletal muscle; BMHO, blue marlin heater organ.

View larger version (17K): [in a new window]   Fig. 2. Western blot analysis with SERCA 1- and SERCA 2-specific antibodies reveals the expression of SERCA 1 in heater cells. Two peptides, one corresponding to amino acids 328-342 of blue marlin SERCA 1B and one corresponding to amino acids 192-205 of rabbit SERCA 2, were used to synthesize isoform-specific Ca2+-ATPase antibodies. The SERCA 1 antibody labeled an approximately 110 kDa band in superior rectus extraocular muscle, heater organ and fast-twitch muscle, while the SERCA 2 antibody labeled atrium, ventricle and slow-twitch muscle. BMSR, blue marlin superior rectus muscle; BMHO, blue marlin heater organ; BMFT, blue marlin fast-twitch skeletal muscle; BMCard, blue marlin cardiac muscle; BFTFT, bluefin tuna fast-twitch skeletal muscle; BFTST, bluefin tuna slow-twitch skeletal muscle; BFTAt, bluefin tuna atrial muscle; BFTVent, bluefin tuna ventricular muscle; YFTFT, yellowfin tuna fast-twitch skeletal muscle; YFTST, yellowfin tuna slow-twitch skeletal muscle.

View larger version (20K): [in a new window]   Fig. 3. Heater organ sarcoplasmic reticulum (SR) vesicles sequester Ca2+ in an ATP-dependent manner. 0.5 mg ml-1 of heater tissue SR vesicles were added to a cuvette containing 1.5 ml transport buffer, 10 µmol l-1 carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) and 10 µmol l-1 oligomycin. The extravesicular Ca2+ concentration was monitored using the fura-2 fluorescence ratio (340 nm/380 nm). Ca2+ uptake was initiated with the addition of 2.5 mmol l-1 MgATP, and sequential additions of 75 nmol CaCl2 were used to load the vesicles. (A) The Ca2+ ionophore A23187 releases the SR-loaded Ca2+. (B) The uptake of Ca2+ is blocked by the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin.

View larger version (18K): [in a new window]   Fig. 4. Heater organ sarcoplasmic reticulum (SR) vesicles release Ca2+ through ryanodine receptor (RyR) channels. (A) After the heater SR vesicles have been loaded with Ca2+, a very small Ca2+ release event can be evoked by the addition of 10 mmol l-1 caffeine and 350µmoll-1 ryanodine. (B) In similarly loaded SR vesicles isolated from toadfish swimbladder fast-twitch muscle, the addition of 10 mmol l-1 caffeine and 350µmoll-1 ryanodine resulted in a large Ca2+ release event. (C) In the absence of an ATP-regenerating system (creatine phosphate and creatine phosphokinase), a much larger release event is evoked in heater tissue by the addition of 10 mmol l-1 caffeine and 350µmoll-1 ryanodine, indicating that the strong presence of the pump in heater tissue limits SR Ca2+ release in this assay.

View larger version (123K): [in a new window]   Fig. 5. Light micrographs of heater tissue labeled with (A) an acetylcholine receptor antibody or (B) rhodamine-conjugated -bungarotoxin. Extensive junctional endplates are present on the surface membrane of heater cells, suggestive of a nervous control of thermogenesis.

View larger version (22K): [in a new window]   Fig. 6. A model of excitation—thermogenic coupling in heater cells. Thermogenesis in heater cells is proposed to occur via depolarization-induced Ca2+ release pathways. Nervous stimulation mediated by acetylcholine receptors (AchR) results in heater cell depolarization and DHPR—RyR1-mediated Ca2+ release. Increased cytoplasmic Ca2+ stimulates Ca2+ transport and ATP turnover by SERCA 1B and mitochondrial influx and efflux pathways. The physiological properties of the RyR1-slow isoform expressed in heater cells may facilitate prolonged channel openings under these conditions (high Ca2+ and the presence of adenine nucleotides) and promote further release of Ca2+ in a `futile' cycle that results in thermogenesis. Abbreviations: T-tubule, transverse-tubule; SR, sarcoplasmic reticulum; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; RyR, ryanodine receptor; DHPR, dihydropyridine receptor.

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