Original Contributions |
From the Departments of Molecular Physiology and Biological Physics (R.E.L., A.P.S., A.V.S.), Anesthesiology (R.E.L.), Internal Medicine (A.P.S.), and Pathology (A.V.S.), University of Virginia Health Sciences Center, Charlottesville; the Department of Biomedical Sciences (G.F.N.), Institute of Medical Science, University of Aberdeen (Scotland), Foresterhill; the Department of Molecular Biology (S.F.), Vanderbilt University Medical Center, Nashville, Tenn; and the Department of Pharmacology (J.A.A.), University of Nevada School of Medicine, Reno.
Correspondence to Ryan E. Lesh, MD, Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, PO Box 10011, Charlottesville, VA 22906-0011. E-mail rel4u{at}elvis.med.virginia.edu
| Abstract |
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Key Words: smooth muscle ryanodine receptor electron microscopy confocal microscopy immunofluorescence
| Introduction |
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The RyR was first isolated from skeletal muscle SR and found to be equivalent to the "foot" structures bridging the T tubules with the terminal cysternae (observed by electron microscopy8 9 )the major site of Ca2+ release in skeletal muscle. Similar "bridging structures" connect the junctional SR with the plasma membrane in smooth muscle,10 but their relationship to RyRs or IP3 receptors has yet to be established. Radioligand binding studies have shown radiolabeled ryanodine binding to the SR fraction of smooth muscle cell homogenates,11 12 13 but the distribution between peripheral (junctional) SR close to the plasmalemma and the central SR has not been previously determined. In the present study, we demonstrate with immunofluorescence and immunoelectron microscopy the presence of ryanodine receptors in both central and peripheral SR of aortic and vas deferens smooth muscle.
| Materials and Methods |
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350 g (obtained
from Hilltop Farms, Scottdale, Pa) were given an overdose of halothane
anesthesia and then exsanguinated by following a protocol
approved by the University of Virginia Animal Experimentation Committee
and in accordance with policies outlined in the Public Health Service
Policy on Humane Care and Use of Laboratory Animals. The thoracic aorta
and vas deferens were dissected free and placed in
HEPES-buffered normal Krebs' solution at 37°C. The connective tissue
was gently removed before fixation. Strips of thoracic aorta were
carefully dissected with microdissection instruments under a dissecting
microscope and then pinned onto dental wax strips at approximately in
vivo length. The psoas muscle was prepared by freeing the muscle from
the surrounding fascia and clamping it at resting length in situ with
Krause muscle biopsy forceps. The clamped portion of the muscle was cut
away from the muscle belly and then handled like the other specimens.
Tissue for confocal microscopy was fixed overnight at 4°C in freshly
prepared 3% paraformaldehyde in 10 mmol/L PBS, pH
7.4. The fixed tissue was cut into
5-mmx8-mm lengths and
cryoprotected in 5% sucrose-PBS (wt/vol), pH 7.4, for 60 minutes at
4°C and then in 15% sucrose-PBS, pH 7.4, at 4°C for an additional
45 to 60 minutes. Blood vessel lumina were filled with Tissue-Tek
O.C.T. compound (Miles Inc) immediately before freezing. All specimens
were rapidly frozen by plunging into Freon-22 subcooled with liquid
N214 and kept in Freon-22 frozen
with liquid N2 until cryosectioning. Tissue samples prepared for immunogold labeling were fixed for 30 minutes in freshly prepared 3% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4) at room temperature and then for 3.5 hours at room temperature in a mixture of 0.2% glutaraldehyde and 3% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4). The tissue was cut into small pieces (<1 mm2) and infiltrated with 2.3 mol/L sucrose in 0.1 mol/L phosphate buffer, pH 7.4, for at least 2 hours. Excess sucrose was removed, and the tissue blocks were mounted on cryopins and rapidly frozen by plunging into liquid N2subcooled Freon-22. Specimens were stored in Freon-22 frozen in liquid N2 until cryosectioning.
Anti-RyR Antibodies
Two preparations of polyclonal antibodies were made in two
different rabbits (antiRyR 81 and antiRyR 82) against a
synthetic peptide that corresponded to the published RyR amino acid
sequence (20 amino acids, 4681 to 4700; C-LEFDGLYITEQPGDDDVKGQ) as
previously described.15 Briefly, the 20amino
acid synthetic oligopeptide was linked to keyhole limpet hemocyanin
(via lysines, using the bifunctional agent
m-maleimidobenzoyl-N-hydroxysuccinimide) injected
into adult rabbits according to the protocol of Gonatas et
al,16 and the antiserum was used to prepare
affinity-purified antibodies. Immunoblots of crude
homogenates from bovine heart, skeletal muscle terminal
cisternae, cultured bovine aortic endothelial cell
membrane preparations, and purified RyRs all showed one band of
reactivity with the resulting antibody.15 This
peptide sequence was selected because of its homology to the heart
(ryr2) and brain (ryr3) ryanodine receptors
(
85% identity). AntiRyR 8 has been found to react with all three
RyR isoforms (L. Jeyakumar and S. Fleischer, unpublished data,
1997).
The specificity of antiRyR 8 antibodies was established by
immunoblotting with membrane preparations of guinea pig
heart, aorta, and vas deferens, prepared by the method of Jones
et al.17 Briefly, tissues were
homogenized in 0.75 mmol/L KCl and 5 mmol/L
histidine and washed twice in the same buffer after
centrifugation at 14 000g. The pellet was
resuspended in 10 mmol/L NaHCO3 and 5
mmol/L histidine and recentrifuged. This process was repeated,
and samples were rehomogenized. The suspensions were
centrifuged again at 14 000g, and the resulting
supernatant was centrifuged at 105 000g for 30
minutes. The pellet was resuspended in 10 mmol/L Tris (pH 7.4),
1 mmol/L EDTA, 1 mmol/L
4-(2-aminoethyl)-benzenesulfonylfluoride, and 84 µmol/L
leupeptin. Proteins from membrane preparations were fractionated by
SDS-PAGE on a 6% gel and transferred to PVDF membranes (Immobilon-P,
Millipore). After blocking in a 10% (wt/vol) solution of nonfat dry
milk/PBST, the membranes were incubated with antiRyR 82 antibody
(diluted 1:660) for 2 hours at room temperature, followed by incubation
in a secondary anti-rabbit IgG coupled to horseradish peroxidase
(Amersham Life Science). Antibody binding to the RyR bands was
visualized using enhanced chemiluminescence (Amersham Life Science)
according to the manufacturer's instructions. Control studies were
performed with antiRyR 82 by preadsorbing the antibody with
synthetic antigen peptide (20-mer) at a molar ratio of
10:1
(antigen/antibody). Antibody and antigen were incubated together for 30
minutes, on ice, before application to tissue sections or blot
membranes.
A murine monoclonal anti-ryanodine antibody (mAb110E), prepared to avian skeletal muscle foot protein polypeptides,18 was also used for both immunohistochemistry and Western blotting. This antibody has been shown to bind selectively to a variety of avian and mammalian RyR isoforms.19
The reactivity of mAb110E for guinea pig RyR isoforms was determined by
Western blotting. Homogenates of guinea pig aorta, ileum,
cardiac muscle, and psoas muscle were size-fractionated by SDS-PAGE on
7.5% gels and transferred to PVDF membranes. Nonspecific
immunoreactive sites on the PVDF membranes were blocked with a solution
of 10% (wt/vol) nonfat dry milk in PBST, pH 7.3, at room temperature
for
1 hour. The membranes were incubated in mAb110E (diluted
1:20 000) in PBST for 3 hours at room temperature, washed in three
changes of PBST (5 minutes each), and then incubated for 1 hour at room
temperature in a peroxidase-conjugated goat anti-mouse IgG (Goldmark
Biologicals) diluted 1:65 000 in PBST. After incubation in the
secondary antibody, the membranes were washed in three changes of PBST
(5 minutes each) and then in PBS (without Tween 20) to remove residual
detergent and developed using an enhanced chemiluminescence protocol
(Amersham Life Sciences) according to the manufacturer's
instructions.
Anti-Vimentin Antibody Binding Studies
A monoclonal anti-vimentin antibody (clone V9, Sigma
Immunochemicals) was used for both immunocytochemical localization of
vimentin in cultured smooth muscle cells and for immunodetection of
vimentin on Western blots. The specificity of this anti-vimentin
antibody has previously been established20 21 ;
the sensitivity of the V9 antibody for vimentin was determined by
Western blotting against guinea pig smooth muscle
homogenates known to contain predominantly vimentin (aorta)
or desmin (vas deferens). Purified pig aorta vimentin (a gift
from Dr David Hartshorne, University of Arizona) was initially included
as a positive control on Western blots.
An established rat aortic smooth muscle cell line (R51393V9) was grown
to subconfluence on glass microscope coverslips (these cells were a
gift from Dr Gary Owens, University of Virginia). The formation of
intermediate filament "cables" was induced in the cultured cells by
the addition of 1 µm colcemid (Sigma) to the growth medium for
24 hours.22 Cells were then removed from culture,
rinsed in PBS, and treated for 2 minutes with methanol precooled to
-20°C. Nonspecific immunoreactive sites were blocked by a 20- to
30-minute incubation of the permeabilized cells in
PBS-ALB, pH 7.4, at room temperature. The cells were then exposed to
either the anti-vimentin antibody (diluted 1:50 [vol/vol] in PBS-ALB)
or to either of the two lots of anti-ryanodine antibodies (diluted 1:50
[vol/vol] in PBS-ALB), as previously described for tissue
sections.15 Primary antibody binding sites were
detected indirectly with an affinity-purified, species-specific,
TRITC-conjugated F(ab')2 fragment (Jackson
ImmunoResearch Laboratories, Inc). Cells were incubated for 1 hour in
secondary F(ab')2 fragment at a final
concentration of
10 µg/mL in PBS-ALB for 1 hour at room
temperature, washed with PBS-ALB, and mounted in a buffered
glycerolcontaining medium for examination in the confocal
microscope.
Immunolabeling for Confocal Microscopy
Specimens frozen in Freon-22 were allowed to warm to -20°C in
the cryochamber and then affixed to a cryosectioning chuck, with
Tissue-Tek O.C.T. compound, in either a longitudinal or transverse
orientation. Cryosectioning was performed on a Leica Frigocut 2800 E
(Leica Instruments GmbH). Sections,
5 to 10 µm thick were cut
and collected on gelatin-coated Superfrost Plus glass microscope slides
(Fisher Scientific). An affinity-purified, species-specific,
TRITC-conjugated F(ab')2 (Jackson ImmunoResearch
Laboratories, Inc) was used for indirect fluorescent labeling.
Sections were preincubated in PBS-ALB to block nonspecific
immunoreactive sites. The solution was aspirated from the slide, and
the sections were incubated with either antiRyR 81 or antiRyR
82 diluted 1:1000 or 1:2000 (vol/vol) in PBS-ALB solution. Primary
antibody was then aspirated from the slide, and the sections were
washed with four changes of PBS-ALB (
250 µL) for 5 minutes each.
Nonspecific immunoreactive sites were blocked for 5 to 10 minutes with
a 5% solution (vol/vol) of preimmune donkey serum diluted in PBS-ALB.
This was aspirated and replaced with the TRITC-labeled
F(ab')2 fragment at a final concentration of
10 µg/mL in PBS-ALB solution. Tissue samples were incubated in the
secondary F(ab')2 fragment for 1 hour, washed as
before with four changes of PBS-ALB, and mounted with
phosphate-buffered glycerol containing antibleach (Ted Pella Inc) or
with PBS containing 100 mg/mL DABCO (Sigma). In several double-labeling
experiments, psoas sections were labeled with both 35 nmol/L
BODIPY-phalloidin (which binds to F-actin, located in the I band) and
antiRyR 81 antibody.
Tissue sections of guinea pig aorta were also labeled with antiRyR 81 antibody that was previously incubated with purified pig aorta vimentin. These competition experiments were performed to adsorb antibodies that could have cross-reacted with vimentin epitopes (see "Results"). AntiRyR 81 was incubated for 30 minutes at room temperature in PBS-ALB containing vimentin at a 10:1 molar ratio of vimentin to RyR 81, and then the antibodies were handled as previously described for tissue labeling.
Confocal images were obtained with a Bio-Rad MRC-1000 Zeiss Axiovert 35 laser scanning confocal imaging system equipped with a krypton-argon laser and an oil-immersion lens (x40; numerical aperture, 1.3) or a water-immersion lens (x40; numerical aperture, 1.2). The laser was fitted with either a blue (excitation, 488 nm) or a yellow (excitation, 568 nm) filter block.
Staining of the SR for Electron Microscopy
Guinea pig aorta and vas deferens smooth muscle were
fixed overnight at 4°C in 2% glutaraldehyde and
postfixed in osmium ferrocyanide to stain selectively the SR network,
as previously described by Nixon et al.6 Samples
embedded in Spurr's resin were cut to
70 nm thickness and imaged at
60 keV on a Philips CM-12 microscope. One-micron-thick sections
("thick" sections) were cut, and images were obtained at 200 keV on
a Philips CM-200 TEM for comparison with the 1-µm-thick
immunofluorescence confocal images.
Immunogold Labeling for Electron Microscopy
Immunogold labeling for electron microscopy was carried out
according to Tokuyasu23 as previously
described.6 Cryosectioning was performed on a
Reichert Ultracut S microtome (Leica Instruments GmbH) at -100°C.
Sections (
100 nm thick) were then transferred from the knife edge
using a wire loop containing a droplet of 2.3 mol/L sucrose in 0.1
mol/L phosphate buffer. The droplet was removed from the chamber,
thawed completely at room temperature, and touched to a Formvar (Ernest
F. Fullam, Inc)coated gold electron microscopy grid (carbon-coated,
glow-discharged). The grid was placed section-side down for up to 3
hours on solidified 2% gelatin in 10 mmol/L PBS at 4°C to
remove the sucrose. The gelatin plate was then warmed to 37°C for 20
minutes until it became liquid, and an equal volume of 50 mmol/L
glycine in PBS was added. After 5 minutes, the grids were removed from
the gelatin and incubated for 5 minutes on a droplet of 50 mmol/L
glycine in PBS. The grids were then transferred to droplets of PBS-ALB
for 5 minutes, followed by a 10-minute incubation in 5% normal
goat serum (Jackson ImmunoResearch Labs, Inc). Sections were
washed in two changes of PBS-ALB and incubated on a droplet of the
primary antibody (antiRyR 81 diluted 1:1000) overnight at 4°C.
Control sections were exposed only to secondary antibody. Grids were
washed in six changes of PBS/BSA for a total of 30 minutes and
incubated for 1 hour with a goat anti-rabbit
F(ab')2 fragment covalently linked to a 1.4-nm
gold particle (diluted 1:100, Nanoprobes, Inc). After 1 hour of
incubation at room temperature with the secondary antibody, grids were
washed once in PBS-ALB and then in three additional changes of PBS for
a total of 20 minutes. Sections were postfixed in PBS with 2%
glutaraldehyde for 10 minutes and washed in three
changes of distilled H2O (total time, 15
minutes). Gold particles were silver-enhanced using an HQ silver
enhancement kit (Nanoprobes, Inc) and then washed in three changes of
distilled deionized H2O. Sections were
simultaneously stained and embedded by incubating them in
1% polyvinylalcohol in a 2% organotungsten stain (Nanoprobes, Inc)
for 10 minutes (modified from Reference 2424 ). Grids were removed from
the droplet with a wire loop, and excess solution was removed by
touching the grid edge to Whatman's No. 50 filter paper and examined
in a Philips CM12 electron microscope at 120 keV.
Gold particles were counted in sections incubated with antiRyR 8 antibody to quantify their location within the cell. Cell profiles were divided into six distinct areas: peripheral SR, central SR, mitochondria, nucleus, cytoplasm, and extracellular space. The number of gold particles within each of these areas was recorded. SR of more than three caveolar lengths from the cell membrane was regarded as central SR.25 Only profiles of whole cells with the entire cell area visible (25 cells for each antibody) were used for particle counting.
| Results |
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60 kD with several lower molecular weight
bands (presumably breakdown products of vimentin). The
anti-vimentin antibody failed to detect any proteins in
homogenates of vas deferens, which is
consistent with reports that 10-nm filaments in nonvascular
smooth muscle (from gut and urogenital sources) are composed of desmin
rather than vimentin.26 Therefore, antiRyR 81
could be used for selectively labeling RyR in the vas deferens,
but not aortic smooth muscle or endothelial cells,
which express vimentin.26 27 28
|
The second antibody, antiRyR 82, was also tested for specificity.
Western blots of membrane preparations from guinea pig vas
deferens, aortic, and cardiac muscle revealed a single
high-molecular-weight band at
400 kD when probed with antiRyR 82
(Fig 1A
). Preadsorption of antiRyR 82 with peptide antigen blocked
the recognition of the
400-kD band found on blots of smooth muscle
membrane fractions from guinea pig aorta, vas deferens, and
cardiac muscle. Two low-molecular-weight bands (
80 and 100 kD) on
blots of aortic smooth muscle membrane preparations presumably
represent degradation products of the ryanodine receptor,
as they were not seen on other immunoblots. AntiRyR 82
bound to the RyR protein from skeletal muscle terminal cisternae but
showed no cross-reactivity with vimentin on dot blots containing
purified pig lens vimentin.
The specificity of a third RyR antibody, mAb110E, was tested on whole
tissue homogenates from guinea pig skeletal muscle (psoas),
cardiac muscle, aorta, and ileum by Western blotting. Because of the
large amount of guinea pig tissue required to prepare membrane
fractions, we determined the reactivity of the mAb110E on whole
homogenates, rather than membrane preparations, of
skeletal, cardiac, and smooth muscle of the aorta and ileum. A single
high-molecular-weight (
400-kD) protein band was present in
skeletal muscle, and a typical doublet was seen in the cardiac lane.
There was weak signal in the same region in the lane containing
homogenate of ileum smooth muscle; however, no signal was
detected in the lane containing aorta homogenate. The
abundant signal in the striated compared with the smooth muscle lanes
(at similar total protein loading) reflects the much higher content of
junctional SR in skeletal and cardiac muscle.
Immunofluorescent Localization of Anti-RyR Binding
Sites
Labeling of skeletal muscle with either antiRyR 81 or
antiRyR 82 produced periodic double rows of punctate
fluorescence (Fig 2
). The
distance from the center of one double row of fluorescence to
the next was
3.0 to 3.6 µm. Double-labeling experiments with
antiRyR 81 and phalloidin-FITC (the latter to identify F-actin in
the I band) established that the punctate fluorescence, from
TRITC-conjugated secondary antibody bound to anti-RyR, was
located at the A-I junction where the T tubules form triad
junctions with the terminal cysternae. Sections labeled with
either antiRyR 81 or antiRyR 82 revealed a similar pattern
of fluorescent signal; however, the double row of
fluorescence was more clearly defined with antiRyR 82 (see
Fig 2
).
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AntiRyR 82 binding was also apparent in longitudinal sections of
thoracic aorta smooth muscle (Fig 3a
and 3b
). Fluorescence was present in both the vascular
endothelial cells, as previously shown with antiRyR
81,15 and in subendothelial
smooth muscle cells that alternate with rows of elastic lamellae in the
media. Fluorescent labeling of cytoplasmic structures was
varied, inhomogeneous, and seen in every cell. Frequently,
on close inspection, the staining pattern was meshlike (arrows in Fig 3a
), suggestive of the SR network. Nuclei were largely void of signal,
but in some cells, there was increased labeling at the nuclear poles in
both endothelial and aortic smooth muscle cells (see
Fig 3a
). Control tissue sections labeled with either
antigen-preadsorbed antibody or TRITC-conjugated donkey anti-rabbit
F(ab')2 revealed no intracellular
fluorescence when imaged under identical conditions (pinhole,
laser intensity) of confocal signal detection.
|
Immunoelectron micrographs of aortic smooth muscle cells labeled with antiRyR 81 revealed that the antiRyR 81 used in our initial experiments,15 but not the antiRyR 82, also labeled (in addition to SR-associated epitopes) intermediate (10-nm) vimentin filaments. In control experiments, similar patterns of antiRyR 81 and anti-vimentin labeling of intermediate filaments were present in cultured rat aortic smooth muscle cells treated with colcemid to induce the formation of intermediate filament cables.22 Sections of thoracic aorta labeled with anti-vimentin antibody showed intense cytoplasmic fluorescence in the endothelial cells and weaker signal in the cytoplasm of the smooth muscle cells. We were unable to adsorb vimentin-binding epitopes in the antiRyR 81 by preincubating the antibody with purified pig lens vimentin before labeling tissue sections.
Sections of thoracic aorta labeled with mAb110E showed patterns
of fluorescence in endothelium and smooth
muscle that were similar to sections labeled with the polyclonal
antiRyR 82. Confocal microscopy with
1-µm optical slices
through the center of a cell showed no significant nuclear labeling
with any of the three RyR antibodies. Control sections exposed only to
secondary antibody showed no significant fluorescent signal
when imaged at similar conditions (pinhole and laser intensity) of
confocal signal collection.
Guinea pig vas deferens cut in transverse orientation and
labeled with antiRyR 82 revealed inhomogeneous
circumferential fluorescence (see Fig 4
). The periphery of some cells showed a
punctate pattern of fluorescence (arrows in Fig 4
), similar to
the predominantly peripheral distribution of SR seen in
osmium ferrocyanidetreated cells (see Fig 6
). When viewed with
similar image-detection parameters, the quantity of
fluorescence seen in these images was significantly less than
in sections of aorta, consistent with the smaller volume of SR
in the vas deferens.
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Electron Microscopy of Aortic and Vas Deferens Smooth Muscle
Postfixed With Osmium Ferrocyanide
Electron microscopy of guinea pig aorta treated with osmium
ferrocyanide showed an extensive network tubular staining throughout
the cytoplasm. As seen in Fig 5
, there is
an interconnecting reticulum that is continuous with the outer nuclear
envelope, extends throughout the cytoplasm frequently encircling the
mitochondria, and forms apparent surface couplings with the plasma
membrane. One-micron-thick sections, imaged at 200 keV to visualize the
same tissue volume imaged with confocal microscopy (see Fig 5c
and 5d
),
showed that the superimposition of the SR tubules in the thick sections
gives rise to a dense, patchy, meshlike network, which, when viewed at
a magnification similar to the fluorescent images (insert, Fig 5d
), resembles the fluorescence
in Fig 3a
and 3b
.
|
Electron micrographs of guinea pig vas deferens postfixed with
osmium ferrocyanide, in contrast to those of the aorta, showed a less
dense reticular network located predominantly at the periphery of the
cells and at the nuclear poles (see Fig 6
). Similar to aortic smooth
muscle, the reticulum is continuous with the outer nuclear envelope and
is present in the cytoplasm. Interestingly, the outer mitochondrial
membranes were often very close (<20 nm) to elements of the SR
network, which at times encircled an entire mitochondrion (arrows in
Fig 6c
).
Immunogold Labeling
In cryosections of vas deferens (which contains no
detectable vimentin) immunolabeled with antiRyR 81,
silver- enhanced gold particles were seen on SR membranes,
predominantly near the plasma membrane (Fig 7A
and 7B
). Occasional immunogold
labeling was also seen in central areas of the cell and on vesicular
membranes of the perinuclear SR (Fig 7C
), consistent with the
sparse central SR in this muscle. AntiRyR 81 did not label
caveolae, mitochondria, nuclei, or the nuclear membrane. The
extracellular space contained only occasional gold particles. Paired
control sections, which were incubated with primary antibody or treated
with preimmune rabbit serum, also contained a negligible number of gold
particles. Attempts to immunogold label cryosections of vas
deferens with antiRyR 82 were unsuccessful, even at low
dilutions of this primary antibody.
|
The number of gold particles counted in each area of the vas
deferens cells labeled with antiRyR 81 is shown in Fig 8
. Not all elements of the SR were
labeled, but gold particles on the SR (central plus
peripheral) accounted for
90% of the total particles
counted. The majority of particles per cell cross section were
peripheral, consistent with the distribution of the
SR. Binding of gold particles to central SR was
50% to 60% of that
found on peripheral SR. The cytoplasm, extracellular space,
mitochondria, and nuclei all contained a much lower density of gold
particles.
|
| Discussion |
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85%
homology with both ryr2 (cardiac)33
and ryr3 (brain and smooth muscle).34
Monoclonal antibody mAb110E recognizes one or more of the RyR isoforms
present in cardiac tissue and brain, although its epitope has not
been mapped.18 Vascular smooth muscle is reported
to contain predominantly ryr334 ; however,
mRNA transcripts for all three isoforms have been identified in various
smooth muscles using reverse transcriptionpolymerase chain
reaction.35 mRNA transcripts for ryr3
and small amounts of ryr2 have been found in aortic smooth
muscle.36 Our results indicate that RyRs are present in guinea pig aorta and vas deferens smooth muscle and are localized to both the central and peripheral SR. The labeling of the triadic region in guinea pig skeletal muscle with antiRyR 8 confirmed previous studies localizing the foot structures (RyR-1) to the terminal cisternae of the SR in the triad (References 37 and 3837 38 and review in Reference 3939 ) and also established the efficacy of these antibodies for immunolabeling. We now show that with the RyR-specific antibodies (antiRyR 82 and mAb110E), labeling occurs at the nuclear poles and in irregular patches in the cytoplasm of endothelial cells, in agreement with our earlier results that were produced with the less specific antiRyR 81.15 These results are consistent with the distribution of the endoplasmic reticulum.
The preponderance of the label at the plasma membrane of vas
deferens cells, the patchy cytosolic distribution of
immunofluorescence in 1-µm-thick optical slices
of aortic smooth muscle labeled with either polyclonal antiRyR 82
or monoclonal mAb110E antibody, and immunoelectron microscopic
analysis of the vas deferens using antiRyR 81 were
consistent with the known distribution of SR. This has been
shown with selective SR staining by osmium ferrocyanide in the aorta
and vas deferens (the present study) and with conventional
electron microscopy of the aorta.25 The SR in
smooth muscle (
5% of the fractional volume of the cytoplasm in the
aorta25 ) forms a continuous network, is
contiguous with the nuclear envelope, frequently associates with
mitochondria, and approaches the plasma membrane to form surface
couplings.6 25 These data strongly suggest that
the location of the RyRs in smooth muscle reflects the different
distribution of SR in the two tissues examined. Furthermore, they also
demonstrate that RyRs are not confined to surface couplings but are
distributed over most of the SR network. Surface couplings in smooth
muscles consist of junctional SR separated from the plasma membrane by
an
18-nm gap traversed by periodic bridging structures. These
electron-dense structures are reminiscent of RyRs localized to "foot
processes" connecting the SR and plasma membranes/T tubules in
skeletal and cardiac muscle.10 40 41 However, on
close examination, the bridging structures are different, in that the
spacing of feet between bridging structures is greater and the density
is less than in striated muscles.42 43
Considering the localization of RyRs to terminal cisternae, it may seem
surprising that in smooth muscle RyRs are present on both central
and peripheral SR. However, this need not imply similar
excitation-contraction coupling mechanisms and raises the possibility
of different mechanisms gating the RyRs in peripheral SR
and central SR, respectively.39 Electron probe
x-ray microanalysis has shown that Ca2+
is sequestered in both central and peripheral SR and that
in norepinephrine-stimulated muscles the
Ca2+ content is decreased, compared with
unstimulated muscles, in both regions.44 45 The
presence of RyRs on the central SR of smooth muscles, which lack
transverse tubules, is also consistent with some findings in
skeletal and cardiac muscle. Dulhunty et al,46
using immunogold labeling techniques, detected RyRs in rat skeletal
muscle not only on the junctional face of the terminal cisternae but
also on the extrajunctional SR, and Jorgensen et
al47 found RyRs in corbular SR, a bulbous
protrusion of cardiac muscle SR containing calsequestrin and
Ca2+, but distant from transverse
tubules.43 They suggested that these receptors
may release Ca2+ directly into the myoplasm
rather than into the triad junction. In rat papillary muscle, Jorgensen
et al47 also demonstrated the presence of
ryanodine receptors in both junctional SR and surface couplings, both
of which also store Ca2+.48
The extended junctional SR of avian cardiac muscle also contains
functional RyRs.49
IP3 receptors are also present in both peripheral and central SR in vas deferens,6 suggesting a structural overlap between the IP3-sensitive Ca2+ pools and the ryanodine-sensitive Ca2+ pools in both types of smooth muscle, as in cerebellar Purkinje neurons.50 Analytical subfractionation has also detected significant colocalization of both IP3 and RyRs in the SR of intestinal smooth muscle.11 Well-controlled double-labeling experiments of the IP3 receptor and RyR at electron microscopic resolution would, however, be necessary to define their proximity.
Although IP3 is thought to be the
physiologically important mediator of
pharmacomechanical Ca2+ in smooth muscle
(reviewed in References 1 and 511 51 ), vascular and other smooth muscles
also possess a caffeine-sensitive intracellular
Ca2+ store that contains sufficient
Ca2+ to produce, when released, tension
development.45 52
Ca2+-induced Ca2+ release
from ryanodine receptors, similar to that seen in
cardiac53 54 and skeletal muscle (reviewed
in Reference 3939 ), also occurs in various smooth
muscles.7 55 56 57 58 Localized domains of high
[Ca2+]i may exist near
surface couplings of junctional SR59 sufficient
to raise local Ca2+ to the
1-µmol/L level
required to trigger Ca2+-induced
Ca2+ release.7 55 60
Caffeine and ryanodine-sensitive Ca2+ spikes
originating from the peripheral SR of vascular smooth
muscle have been observed and are, in many cells, coupled to the
activation of transmembrane cation currents.61
Ca2+ sparks contribute to relaxation of
cerebrovascular smooth muscle via activation of a plasma membrane
Ca2+-sensitive K+ channel
and plasma membrane
hyperpolarization.62
Activation of Ca2+ release through
peripheral RyRs could, then, be gated by dihydropyridine
receptors, as in skeletal muscle, or by Ca2+, as
in cardiac muscle cells.63 Activation of RyRs in
the central SR, however, would require a diffusible second messenger
like Ca2+ or cADP-ribose.64
During excitation-contraction coupling, other factors, such as
phosphorylation65 66 and/or
modulators of Ca2+ release from the RyRs,
including Mg2+, Ca2+, pH,
calmodulin, and adenine
nucleotides,67 may also alter the
physiological responsiveness of these channels in
vivo.
| Selected Abbreviations and Acronyms |
|---|
|
| Acknowledgments |
|---|
| Footnotes |
|---|
Received March 20, 1997; accepted November 7, 1997.
| References |
|---|
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