Characterization of ryanodine receptor and Ca2+-ATPase isoforms in the thermogenic heater organ of blue marlin (Makaira nigricans)
Stanford University, Hopkins Marine Station, Pacific Grove, CA 93950,
USA
Present address: Occidental College, Los Angeles, CA 90041, USA
* Author for correspondence (e-mail: morriss{at}stanford.edu)
Accepted 21 November 2002
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Summary |
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Key words: ryanodine receptor, RyR, Ca2+-ATPase, SERCA, thermogenesis, heater organ, marlin, Makaira nigricans
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Introduction |
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Heater organs are derived from modified extraocular muscle cells that have
been optimized for heat production rather than contractility. Heater cells
have lost organized myofilaments (Block and
Franzini-Armstrong, 1988;
Tullis and Block, 1997
) but
have retained an extensive transverse-tubule (T-tubule) network and
sarcoplasmic reticulum (SR) complete with triads (Block et al.,
1988a
,
b
,
1994
). Electron microscopy
studies revealed that as much as 60% of the heater cell in billfishes is
composed of tightly packed mitochondria
(Tullis et al., 1991
).
Biochemical studies have shown that heater cells have an exceptional aerobic
capacity (Ballantyne et al.,
1992
; Tullis et al.,
1991
) and a tight coupling of oxidative phosphorylation and
electron transport in the blue marlin
(O'Brien and Block, 1996
).
However, Ballantyne et al.
(1992
) demonstrated that high
millimolar [Ca2+] uncoupled swordfish mitochondria
(Ballantyne et al., 1992
).
Uncoupling proteins have not been found in heater tissue
(Block, 1986
). The presence of
an extensive T-tubule and SR network in close proximity to coupled
mitochondria indicates that a Ca2+-dependent process may underlie
thermogenesis in heater cells (Block and
Franzini-Armstrong, 1988
). However, due to the challenges of
conducting in vivo work on large pelagic fishes, this hypothesis has
never been fully tested. The present study provides an in vitro assay
that indicates that blue marlin heater tissue has exceptional Ca2+
uptake activity with rates comparable with the superfast-contracting toadfish
swimbladder muscle. Furthermore, molecular and physiological studies indicate
that the extraocular muscle fibers and the heater cells express
physiologically unique isoforms of the SR ryanodine receptor (RyR1-slow) and
Ca2+-ATPase (SERCA 1B), which may predispose these cells for
Ca2+ cycling and thermogenesis. In addition, innervation of heater
tissue by a branch of the oculomotor neuron and the presence of extensive
clusters of acetylcholine receptors (AchR) on the heater plasma membrane
indicate that thermogenesis may be under neural control.
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Materials and methods |
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Ca2+-ATPase antibody production and purification
Two peptides, one corresponding to amino acids 328-342 of blue marlin SERCA
1 (Londraville et al., 2000)
and one corresponding to amino acids 192-205 of rabbit SERCA 2
(Brandl et al., 1986
), were
synthesized by Research Genetics Inc. (Huntsville, AL, USA) and used to
immunize four rabbits for polyclonal antibody production. Serum immunoglobulin
G (IgG) purification was performed using ImmunoPure Affinity PakTM
protein A columns following the instructions provided by the manufacturer
(Pierce, Rockford, IL, USA). Two affinity columns were made by immobilization
of 5.0 mg of the synthesized peptides provided by Research Genetics Inc. using
an EDC [1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide]/Diaminodipropylamine
Immobilization Kit (Pierce, Rockford, IL, USA). 1 ml of the IgG fractions from
the protein A columns followed by 2.0 ml of PBS (phosphate-buffered saline)
were applied to the appropriate affinity column and incubated for 1 h at room
temperature. The columns were washed with an additional 14 ml of PBS, after
which the SERCA (sarco/endoplasmic reticulum Ca2+-ATPase)
antibodies were eluted with 0.1 mol l-1 glycine, pH 3.0. 1 ml
fractions were collected and their absorbance at 280 nm was monitored to
determine the presence of antibody. Fractions with the highest absorbance were
pooled, adjusted to neutral pH and stored at -80°C for later use. This
protocol resulted in the production and purification of two SERCA antibodies,
one able to recognize SERCA 1 and the other able to recognize SERCA 2.
Immunohistochemistry and -bungarotoxin labeling
Immunohistochemical analysis of sectioned heater tissue was performed as
previously described (Block et al.,
1994; Tullis and Block,
1996
). Briefly, 10 µm thick slices of frozen heater tissue were
mounted on slides and incubated in PBS containing 0.5 mmol l-1
ascorbic acid and 0.05 mmol l-1
N,N,N',N'-tetramethyl-p-phenylenediamine, pH 7.5
for 40 min to reduce auto-fluorescence. Sections were then incubated in PBS
supplemented with 10% goat serum and 0.3% Triton-X for 30 min followed by an
overnight incubation in AchR antibody diluted 1:200 in PBS or 10 µg
ml-1 of rhodamine-conjugated
-bungarotoxin in PBS at
4°C. Sections were washed for 3x10 min in PBS. Antibody-labeled
sections were incubated for 30 min at room temperature in a
fluorescein-conjugated secondary antibody at 1:50 dilution in PBS and then
washed for 3x10 min in PBS.
-bungarotoxin-labeled sections were
washed for 3x10 min in PBS. Sections were mounted in 4% n-propyl
gallate, 80% glycerol, 20 mmol l-1 Tris, pH 9.0 and examined using
a Zeiss Axioplot microscope (Carl Zeiss Inc., Thornwood, NY, USA) equipped
with an epifluorescence attachment. Control sections were incubated with
secondary antibody only or with 100 µg ml-1 unlabeled
-bungarotoxin prior to rhodamine-conjugated toxin.
Ca2+ fluorimetry
Sarcoplasmic reticulum Ca2+ uptake and release was measured
using the Ca2+-sensitive dye fura-2 and a Shimadzu RF 5301
spectrofluorophotometer (Shimadzu Scientific Instruments, Kyoto, Japan). SR
microsomes were added at a final concentration of 0.5 mg ml-1 to a
cuvette containing 2.0 ml of transport buffer [95.0 mmol l-1 KCl,
20.0 mmol l-1 H-Mops, 7.5 mmol l-1
Na4P2O7 (sodium pyrophosphate), 5.0 mmol
l-1 creatine phosphate, 0.01 mg ml-1 creatine
phosphokinase and 1.5 µmol l-1 fura-2 (potassium salt), pH 7.0].
Extravesicular Ca2+ was monitored as the ratio of fura-2
fluorescence emission intensity at excitation wavelengths of 340 nm and 380
nm. Ca2+ uptake was initiated with the addition of 2.5 mmol
l-1 MgATP and allowed to proceed to steady state. Sequential 75
nmol additions of CaCl2 were made to the cuvette to load the
vesicles to a filling capacity of 0.8-1.0 µmol Ca2+
mg-1 protein. 10 mmol l-1 caffeine and 350 µmol
l-1 ryanodine were added to stimulate Ca2+ release. In
some experiments, the activity of the SERCA pumps was reduced by the omission
of creatine phosphate and creatine phosphokinase from the transport
buffer.
Gel electrophoresis and western blot analysis
Heater tissue SR microsomes were run on 7.5% SDS (sodium dodecyl
sulfate)polyacrylamide gels according to the method of Laemmli
(1970). Gels were blotted onto
PVDF (polyvinylidine fluoride) membranes that were subsequently blocked in 5%
non-fat milk, 0.2% tween in PBS for >1.5 h. Blots were incubated overnight
at 4°C in primary antibody diluted 1:500 in PBS. Blots were washed three
times in PBS and subsequently incubated with an alkaline
phosphatase-conjugated goat anti-rabbit secondary antibody diluted 1:1000 in
PBS for 1 h. Blots were developed with BCIP/NBT (5-bromo-4-chloro-3-indolyl
phosphate/nitro blue tetrazolium).
Ribonuclease protection assays (RPAs)
RNase protection assays were performed as previously described
(Franck et al., 1998).
Briefly, two ryanodine receptor antisense probes were constructed: one that
hybridized strictly to transcripts of RyR1-slow, an RyR specific to
slow-twitch muscle, and one that hybridized strictly to transcripts of
RyR1-fast, an RyR specific to fast-twitch muscle. The RyR1-slow probe was
synthesized from a subcloned region amplified from a blue marlin eye muscle
RyR clone [nucleotides 4075-4315 in the blue marlin RyR1-slow open reading
frame (ORF)], while the RyR1-fast probe was synthesized from a subcloned
region amplified from a toadfish swimbladder muscle RyR clone (nucleotides
13737-14130 in the blue marlin RyR1-slow ORF). Both antisense probes were
synthesized from EcoRI linearized plasmids with T7 RNA polymerase and
[32P]dUTP according to the Ambion Maxiscript T7/T3 in
vitro transcription kit protocol (Ambion Inc., Austin, TX, USA). Total
RNA used for the RPAs (20 µg assay-1) was prepared using Tri
Reagent (Molecular Research Center Inc., Cincinnati, OH, USA) with the RPA
performed using the Ambion Direct Protect RPA kit (Ambion Inc.). All
hybridizations were performed at 37°C. Samples were separated on a 6%
sequencing gel that was dried and exposed to x-ray film for 24-72 h at
-80°C.
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Results |
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Identification and characterization of heater cell SERCA
Western blotting with antibodies constructed to specifically label SERCA 1
or SERCA 2 epitopes indicates that heater cells express the SERCA 1 isoform of
the Ca2+-ATPase (Fig.
2). The SERCA 1-specific antibody was raised to amino acids
328-342 of blue marlin SERCA 1
(Londraville et al., 2000) and
labeled SR vesicles from blue marlin and bluefin tuna fast-twitch swimming
muscle, blue marlin extraocular muscle and heater organ but did not detect the
SERCA 2 isoforms in bluefin heart or slow-twitch muscle. Conversely, the SERCA
2-specific antibody, raised to amino acids 192-205 of rabbit SERCA 2, did not
label heater organ SR vesicles but positively labeled heart and slow-twitch
muscle. The complete cDNA sequence for the SR Ca2+-ATPase (SERCA)
has been cloned from blue marlin extraocular muscle
(Londraville et al., 2000
).
The sequence codes for 996 amino acids and corresponds to a neonatal isoform
of Ca2+-ATPase found in mammals (SERCA 1B). The marlin SERCA 1B
sequence is identical to marlin SERCA 1A expressed in marlin fast-twitch
muscle except for an additional five amino acids at the carboxyl terminus.
RNase protection assays using probes constructed to hybridize to SERCA 1B
reveal that heater tissue expresses SERCA 1B
(Londraville et al., 2000
).
The western blot results show that heater cells express the SERCA 1 protein
and corroborate the molecular work of Londraville et al.
(2000
).
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Characterization of Ca2+ uptake and release by heater cell
SR
The Ca2+-sensitive dye fura-2 was used to monitor the uptake and
release of Ca2+ by blue marlin heater SR vesicles. The addition of
2.5 mmol l-1 MgATP stimulated Ca2+ uptake by heater SR
vesicles, evident as a decrease in the ratio of the fura-2 fluorescence
(Fig. 3A). Sequential additions
of 75 nmol of CaCl2 actively loaded the vesicles to an apparent
steady-state filling capacity of approximately 0.8-1.0 µmol
Ca2+mg-1 protein. Once Ca2+ has been loaded
into the SR lumen, it can be rapidly released by the addition of the
Ca2+ ionophore A23187. The sequestration of Ca2+ by
heater SR is completely blocked by the addition of 10 µmol l-1
thapsigargin, indicating the active nature of the loading phase
(Fig. 3B). Ca2+
uptake rates for blue marlin SR vesicles were 1.52±0.36 nmol
Ca2+ mg-1 s-1 for uptake of the first-added
Ca2+ bolus and declined to 0.83±0.19 nmol Ca2+
mg-1 s-1 for uptake of the fifth Ca2+
addition. A slowing of the uptake rate can be expected as the vesicles become
progressively filled. These uptake rates are comparable with rates of initial
Ca2+ uptake displayed by toadfish swimbladder muscle SR vesicles
(1.41±0.31 nmol Ca2+mg-1s-1;
Fig. 4B) but are considerably
faster than the 0.69 nmol Ca2+mg-1s-1 uptake
rate observed in fast-twitch skeletal muscle (data not shown).
|
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Caffeine and ryanodine are two agonists of RyRs commonly used to release Ca2+ from SR stores. Surprisingly, the addition of caffeine and ryanodine to Ca2+-loaded blue marlin heater SR vesicles caused little release (Fig. 4A). An identical experiment using fast-twitch toadfish swimbladder muscle SR vesicles, which sequester Ca2+ at a similar rate and to a similar degree as heater tissue SR, resulted in a large proportion of SR Ca2+ released upon stimulation by caffeine and ryanodine (Fig. 4B). One explanation for the apparent lack of significant Ca2+ release in heater SR vesicles is that the release is masked by the high activity of the SERCA pump. This hypothesis is supported by the fact that omission of the creatine phosphate/creatine phosphokinase ATP regenerating system, thus limiting the pump's access to ATP, causes a much larger Ca2+ release event in response to RyR agonists (Fig. 4C). However, even with the SERCA pump activity constrained, the Ca2+ released from heater tissue SR is still less than that from swimbladder muscle SR (Fig. 4C). This suggests that other factors, such as the distinct RyR isoforms in heater and swimbladder muscle (RyR1-slow vs RyR1-fast), may contribute to the differences in caffeine- and ryanodine-evoked Ca2+ release in the two tissues.
Heater organ `endplates'
Heater organs are located beneath the brain, bilaterally situated between
the two superior rectus extraocular muscles. As seen during dissections, a
branch of the oculomotor nerve courses through the heater organ and is
suggestive of a neuronal control of heater organ thermogenesis. To examine
this further, we used immunohistochemistry to identify whether acetylcholine
receptors (AchR) were present along the surface of the heater cells. Using two
methodologies (antibodies with epitopes specific to the AchR or
rhodamine-conjugated -bungarotoxin), we have identified large clusters
and extensive labeling of AchR on the surface of heater cells
(Fig. 5). The presence of these
`endplate-like' labels is indicative of significant junctional membranes,
which may function to couple heater cells and oculomotor nerve endings
electrically in a fashion analogous to the motor endplates of skeletal muscle,
making excitationthermogenic coupling possible.
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Discussion |
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Thermogenesis in fish heater organs occurs in modified extraocular muscle
cells that have lost much of their organized contractile apparatus but express
a high content of mitochondria and SR. Heater cells are innervated by a branch
of the oculomotor nerve (Block,
1986), and, in this study, we have demonstrated the presence of
large clusters of AchR on the plasma membrane, indicative of an endplate-like
structure. The presence of AchR is consistent with a thermogenic mechanism
involving the nervous depolarization of heater cells. The first step in an
excitationthermogenic coupling mechanism probably involves the
transmission of action potentials down the oculomotor neuron, resulting in
release of Ach and depolarization of the heater endplate (see
Fig. 6). Once stimulated,
heater cell depolarization would lead to Ca2+ release from the SR
in much the same manner as it occurs in skeletal muscle.
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Previous studies have established an extensive T-tubule system in heater
cells using intermediate voltage electron microscopy and Golgi labeling
(Block and Franzini-Armstrong,
1988). Junctional complexes between the T-tubule and SR membrane
systems have also been identified (Block et
al., 1988b
). Depolarization of the T-tubule membrane would lead to
a conformational change in the voltage-sensitive dihydropyridine receptors
(DHPR) or L-type Ca2+ channels. To date, no antibodies have been
identified that crossreact with the fish DHPR isoform, and identification and
localization of the DHPR expressed in heater cells has yet to be resolved.
Depolarization of the T-tubule membrane would induce the opening of the RyR,
possibly through a physical mechanical coupling between DHPR and RyR. A direct
mechanical connection between DHPRs and RyR1s is the prevailing hypothesis by
which these two proteins interact in skeletal muscle. However, a mechanism
involving Ca2+-induced Ca2+ release in which RyRs are
opened by the inward Ca2+ current through the DHPR cannot be ruled
out.
In the present study, we have shown that heater tissue expresses RyR1-slow,
an RyR isoform that is also expressed in fish slow-twitch muscle and displays
several physiological properties that may promote thermogenesis. RyR1-slow has
been isolated and characterized from tuna slow-twitch muscle and compared with
RyR1-fast from tuna fast-twitch and toadfish swimbladder muscle
(Morrissette et al., 2000).
The most important finding of this comparative analysis is that adenine
nucleotides attenuate the Ca2+-dependent inhibition of RyR1-slow
but have little effect on the Ca2+-dependent inhibition of
RyR1-fast. When cytoplasmic [Ca2+] rises, most skeletal muscle RyR
channels close; however, in the presence of adenine nucleotides, RyR1-slow
remains open. This property may contribute to a prolonged release of
Ca2+ from the SR in heater cells. Prolonged release of
Ca2+ would promote thermogenesis. Remarkably, the physiological
characteristics of the naturally expressed heater RyR resembles in many
aspects the physiological properties exhibited by the mutated RyR responsible
for the lethal muscular disease malignant hyperthermia (MH). MH is induced by
clinical doses of general anesthetics that stimulate prolonged Ca2+
release due to a mutated RyR (Loke and
MacLennan, 1998
; Mickelson and
Louis, 1996
). MH patients develop a condition characterized by
increased muscle metabolism coupled with muscle rigidity and massive
hyperthermia. The physiological properties of RyR1-slow would promote
prolonged channel opening and Ca2+ release in heater tissue during
thermogenic episodes. The increase in cytoplasmic Ca2+ would
stimulate Ca2+ transport and ATP turnover by the
Ca2+-ATPase pump and mitochondrial influx and efflux pathways that
would consume oxidative energy and promote thermogenesis. The high activity of
the Ca2+-ATPase would attempt to restore cytoplasmic
Ca2+ to resting values; however, the prolonged open state of
RyR1-slow could create a continuous Ca2+ `leak pathway' from the SR
and increase the rate of ATP hydrolysis and heat production.
In the present study, the presence of a SERCA 1 protein in heater tissue is
established by western blots. This is consistent with Londraville et al.
(2000), who used RNase
protection assays to show that heater tissue expresses SERCA 1B, the neonatal
isoform of the Ca2+ pump. However, it is still uncertain whether
heater cells may also express the SERCA 1A isoform. Tullis and Block
(1996
) amplified a PCR product
that indicated that a SERCA 1A isoform was present in heater cells; however,
it was unclear whether the amplified product was from the heater or muscle
cellular component of the tissue (Tullis
and Block, 1996
). Electron microscopy of heater tissue has shown
that muscle fibers expressing a normal muscle cell architecture are often in
close association with the heater cell phenotype. In situ
hybridization studies are needed to definitively resolve the expression
pattern of SERCA 1 isoforms in heater cells. Regardless of the expression
pattern of SERCA 1 isoforms, the data in the present study allow the
characterization of the physiological properties of the heater SERCA
isoform(s). In identical microsomal preparations, heater vesicles sequester
Ca2+ at a rate comparable with toadfish swimbladder muscle, one of
the fastest contracting vertebrate muscles
(Rome, 1999
), and more than
2-fold faster than fast-twitch swimming muscle. This high rate of pump
activity would clearly be advantageous in an excitation thermogenic
coupling process, enhancing ATP turnover and oxidative phosphorylation. The
extent to which the high rate of Ca2+ uptake in heater tissue is
due to the individual properties of the isoforms expressed, or to increased
expression of pump proteins, remains to be seen. Currently, experiments are
underway to further characterize the rate of ATP hydrolysis and the expression
patterns of heater SERCA in a variety of billfish heater cell phenotypes.
In summary, the data indicate that Istiophorid billfish, represented by the blue marlin, have the membrane components to initiate nervous depolarization of heater cells. The resulting depolarization would result in heat production via RyR1-slow mediated Ca2+ release and SERCA 1-mediated Ca2+ reuptake by the SR. The cycling of Ca2+ between the SR and cytoplasm would increase substrate oxidation to fuel the process as well as generate additional heat. The RyR and SERCA isoforms expressed in heater tissue are unique and their physiological properties probably contribute to the heat production process. In these fish species, the presence of slow-twitch fibers close to the brain and eyes probably provides the raw material for the evolution of the heater phenotype.
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Acknowledgments |
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References |
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