Localization of Ryanodine Receptors in Smooth Muscle
Ryan E. Lesh,✳ Graeme F. Nixon,✳ Sidney Fleischer, Judith A. Airey,
Andrew P. Somlyo, Avril V. Somlyo
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Abstract—The ryanodine receptor (RyR) in aortic and vas deferens smooth muscle was localized using immunofluorescence
confocal microscopy and immunoelectron microscopy. Indirect immunofluorescent labeling of aortic smooth muscle with
anti-RyR antibodies showed a patchy network-like staining pattern throughout the cell cytoplasm, excluding nuclei, in
aortic smooth muscle and localized predominantly to the cell periphery in the vas deferens. This distribution is consistent
with that of the sarcoplasmic reticulum (SR) network, as demonstrated by electron micrographs of osmium ferrocyanide–
stained SR in the two smooth muscles. Immunoelectron microscopy of vas deferens smooth muscle showed anti-RyR
antibodies localized to both the sparse central and predominant peripheral SR elements. We conclude that RyR–Ca21release channels are present in both the peripheral and central SR in aortic and vas deferens smooth muscle. This
distribution is consistent with the possibility that both regions are release sites, as indicated by results of electron probe
analysis, which show a decrease in the Ca21 content of both peripheral and internal SR in stimulated smooth muscles. The
complex distribution of inositol 1,4,5-trisphosphate and ryanodine receptors (present study) is compatible with their
proposed roles as agonist-induced Ca21-release channels and origins of Ca21 sparks, Ca21 oscillations, and Ca21 waves.
(Circ Res. 1998;82:175-185.)
Key Words: smooth muscle n ryanodine receptor n electron microscopy n confocal microscopy
immunofluorescence
T
he primary, though not sole, mechanism of smooth muscle
activation is an increase in [Ca21]i, whether the stimulus for
contraction is through a voltage-independent pathway (pharmacomechanical coupling) and/or membrane depolarization
(electromechanical coupling) (reviewed in Reference 1). Increases in [Ca21]i can result from either the trans–plasma
membrane flux of Ca21 through plasma membrane Ca21
channels or the release of Ca21 from intracellular stores, with
the relative contribution from these two Ca21 pools varying
with different smooth muscles and stimuli.1 Ca21 is stored
intracellularly in the SR, which contains at least two types of
Ca21-release channels: the IP3 receptor/Ca21-release channel
and the RyR/Ca21-induced Ca21-release channel. The IP3
receptor is thought to be the major channel mediating pharmacomechanical coupling in smooth muscle2–5 and has been
localized to both the junctional and central SR6; however,
functional studies also provide evidence of a caffeine-releasable
ryanodine-sensitive Ca21 pool in smooth muscle3,7 that, as in
cardiac muscle, may be subject to a Ca21-induced Ca21-release
mechanism triggered by the trans–plasma membrane Ca21
current.
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 Ca21 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,11–13 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
Tissue Preparation
Adult male guinea pigs (Hartley) weighing '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 HEPESbuffered normal Krebs’ solution at 37°C. The connective tissue was
Received March 20, 1997; accepted November 7, 1997.
✳
Both authors contributed equally to this study.
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 [email protected]
© 1998 American Heart Association, Inc.
175
176
Ryanodine Receptors in Smooth Muscle
Selected Abbreviations and Acronyms
IP3 5 inositol 1,4,5-trisphosphate
mAb (with number) 5 monoclonal antibody
PBS-ALB 5 PBS containing 3% bovine serum albumin
PBST 5 PBS with 0.05% Tween 20
PVDF 5 polyvinylidene fluoride
RyR 5 ryanodine receptor
SR 5 sarcoplasmic reticulum
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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-mm38-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 N2–subcooled
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 (anti–RyR 8 –1 and anti–RyR 8 –2) 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 20 –amino 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). Anti–RyR 8 has been found to
react with all three RyR isoforms (L. Jeyakumar and S. Fleischer,
unpublished data, 1997).
The specificity of anti–RyR 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-(2aminoethyl)-benzenesulfonylfluoride, and 84 mmol/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 anti–RyR 8 –2
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 anti–RyR 8 –2 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 mm 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
'220°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(ab9)2 fragment (Jackson ImmunoResearch Laboratories, Inc). Cells were incubated for 1 hour in secondary F(ab9)2 fragment at a final concentration
of '10 mg/mL in PBS-ALB for 1 hour at room temperature, washed
with PBS-ALB, and mounted in a buffered glycerol– containing
medium for examination in the confocal microscope.
Immunolabeling for Confocal Microscopy
Specimens frozen in Freon-22 were allowed to warm to 220°C in the
cryochamber and then affixed to a cryosectioning chuck, with
Lesh et al
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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 mm thick were cut
and collected on gelatin-coated Superfrost Plus glass microscope slides
(Fisher Scientific). An affinity-purified, species-specific, TRITCconjugated F(ab9)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 anti–RyR 8 –1 or anti–RyR 8 –2 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 mL) 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(ab9)2 fragment at a
final concentration of '10 mg/mL in PBS-ALB solution. Tissue
samples were incubated in the secondary F(ab9)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 anti–RyR 8 –1 antibody.
Tissue sections of guinea pig aorta were also labeled with anti–RyR
8 –1 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”). Anti–RyR 8 –1 was incubated for 30 minutes at room
temperature in PBS-ALB containing vimentin at a 10:1 molar ratio of
vimentin to RyR 8 –1, 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 (340; numerical
aperture, 1.3) or a water-immersion lens (340; 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-micronthick sections (“thick” sections) were cut, and images were obtained
at 200 keV on a Philips CM-200 TEM for comparison with the
1-mm-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 2100°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
(anti–RyR 8 –1 diluted 1:1000) overnight at 4°C. Control sections
were exposed only to secondary antibody. Grids were washed in six
177
changes of PBS/BSA for a total of 30 minutes and incubated for 1
hour with a goat anti-rabbit F(ab9)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 silverenhanced 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 24). 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 anti–RyR
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
Anti-RyR Antibody Specificity
The specificity of anti–RyR 8 –1 for the ryanodine receptor
has previously been determined15 using immunoblots of purified skeletal RyR, skeletal muscle terminal cisternae, and crude
membrane preparations of rabbit skeletal and cardiac muscle
and bovine endothelial cells. However, in the course of
subsequent immunoelectron microscopic experiments, later
preparations of anti–RyR 8 –1 were found to also label, in
addition to SR, intermediate filaments. Therefore, Western
blot and dot blot membranes containing purified vimentin (the
major intermediate filament protein in aortic smooth muscle)
and RyR were probed with anti–RyR 8 –1; this antibody
bound to both RyRs and vimentin. To explore this crossreactivity further, Western blots of cell homogenates of aorta
and vas deferens were probed with anti-vimentin antibody (Fig
1B). Aortic smooth muscle proteins showed one band of
reactivity at '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, anti–RyR 8 –1 could be used for selectively
labeling RyR in the vas deferens, but not aortic smooth muscle
or endothelial cells, which express vimentin.26 –28
The second antibody, anti–RyR 8 –2, 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 anti–RyR 8 –2 (Fig 1A). Preadsorption of anti–RyR 8 –2
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. Anti–RyR 8 –2
178
Ryanodine Receptors in Smooth Muscle
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.
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Figure 1. A, Western blot of membrane preparations from
guinea pig cardiac (C), vas deferens (Vas), and aortic (Ao)
smooth muscle. Membrane preparations were size-fractionated
by SDS-PAGE on 6% gels, transferred to PVDF membranes,
and blotted with anti–RyR 8 –2. These blots demonstrate the
presence of immunoreactive proteins in both Vas and Ao
smooth muscle membrane preparations. B, Western blot analysis of homogenates from guinea pig Vas and Ao smooth muscle. The homogenates were fractionated by SDS-PAGE, transferred to PVDF membranes, and probed with anti-vimentin
antibody. Vimentin is present in the Ao homogenates but absent
in the Vas.
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
Immunofluorescent Localization of Anti-RyR
Binding Sites
Labeling of skeletal muscle with either anti–RyR 8 –1 or
anti–RyR 8 –2 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
mm. Double-labeling experiments with anti–RyR 8 –1 and
phalloidin-FITC (the latter to identify F-actin in the I band)
established that the punctate fluorescence, from TRITCconjugated 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 anti–RyR 8–1 or anti–RyR 8–2 revealed a similar pattern
of fluorescent signal; however, the double row of fluorescence
was more clearly defined with anti–RyR 8–2 (see Fig 2).
Anti–RyR 8 –2 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 anti–RyR 8 –1,15 and in subendothelial smooth muscle cells that alternate with rows of elastic
Figure 2. Photomicrograph of guinea pig psoas muscle labeled with either anti–RyR 8 –1 (left panel) or anti–RyR 8 –2 (right panel) and
then a TRITC-conjugated secondary antibody. Both images show the presence of anti-RyR antibody binding sites in skeletal muscle
and confirm previous studies demonstrating the presence of RyRs in skeletal muscle at the A-I interface. Labeling experiments of skeletal muscle consistently demonstrated double rows of punctate fluorescence separated from each other by '3.0 to 3.6 mm.
Lesh et al
179
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Figure 3. a, Confocal photomicrograph
of guinea pig aorta labeled with anti–
RyR 8 –2 and then with a TRITC-conjugated secondary antibody. There is
cytoplasmic labeling of both endothelial
and aortic smooth muscle cells, and
the label correlates with the distribution
of the SR in both cell types. Arrows
indicate a meshlike staining pattern in
two separate cells, suggestive of the
SR network. b, Higher magnification
view of guinea pig aorta showing similar patterns of immunolabeling with
anti–RyR 8 –2.
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 TRITCconjugated donkey anti-rabbit F(ab9)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 anti–RyR 8 –1 revealed that the anti–RyR 8 –1
used in our initial experiments,15 but not the anti–RyR 8 –2,
also labeled (in addition to SR-associated epitopes) intermediate (10-nm) vimentin filaments. In control experiments, similar
patterns of anti–RyR 8 –1 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 anti–RyR
8 –1 by preincubating the antibody with purified pig lens
vimentin before labeling tissue sections.
180
Ryanodine Receptors in Smooth Muscle
cent 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).
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Figure 4. Confocal photomicrograph of guinea pig vas deferens
sectioned transversely and labeled with anti–RyR 8 –2 and Cy3conjugated anti-rabbit secondary antibody. Cell borders and cell
nuclei were identified in these specimens by differential interference contrast microscopy. However, because differential interference contrast microscopy is not confocal, we cannot exclude
the possibility that the smaller ('3-mm-diameter) fluorescence
profiles were due to perinuclear staining rather than labeling at
the periphery of tapered cells in transverse sections. Asterisks
are placed at the centers of selected cells. Arrows show punctate fluorescence at the cell periphery.
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 anti–RyR
8 –2. Confocal microscopy with '1-mm 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 anti–RyR 8 –2 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 ferrocyanide–treated 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.
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-micronthick 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 fluores-
Immunogold Labeling
In cryosections of vas deferens (which contains no detectable
vimentin) immunolabeled with anti–RyR 8 –1, silverenhanced 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.
Anti–RyR 8 –1 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 anti–RyR 8 –2 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 anti–RyR 8 –1 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
Cloning studies have identified a family of RyRs encoded by
three distinct genes (ryr1, ryr2, and ryr3).29 –31 The peptide
sequence used to generate anti–RyR 8 (C-LEFDGLYITEQPGDDDVKGQ) is a skeletal muscle sequence (ryr1)32 that
shares '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
transcription–polymerase 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 anti–RyR 8 con-
Lesh et al
181
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Figure 5. Electron micrograph of guinea pig aorta treated with osmium ferrocyanide to selectively stain the sarcoplasmic and endoplasmic reticulum, showing an extensive network of reticulum throughout the cytoplasm. a, A 70-nm-thick section showing portions of
two longitudinally oriented guinea pig smooth muscle cells lying between elastic lamellae (el). The densely stained reticulum is continuous (small arrows) with the nuclear envelope and extends through the cytoplasm to the plasma membrane, forming surface couplings
(large arrows). Nuc indicates nucleus. b, Obliquely oriented 70-nm-thick section of an aortic cell showing tubules of reticulum in the
central regions of the cell extending to the periphery, forming surface couplings with the plasma membrane (small arrows). Mitochondria (m) are frequently surrounded by closely apposed elements of reticulum. c and d, Longitudinal views from the same osmium
ferrocyanide–stained blocks of aorta shown in panels a and b, but in this case, sections were cut at a 1-mm thickness for comparison
with the 1-mm optical sections used for the immunofluorescence studies. Note the high density of reticulum membranes, which appear
connected throughout the cell and surround a cluster of mitochondria (m) in panel c. The image of the cells in panel d has been
reduced to a size comparable to that shown in the confocal immunofluorescence images. Note that immunolabeling of ryanodine
receptors associated with this dense complex network of reticulum could give rise to the patchy meshlike staining seen in the confocal
fluorescence images.
182
Ryanodine Receptors in Smooth Muscle
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Figure 6. Electron micrographs of two transverse sections (a and b) and a longitudinal thin section (c) from a guinea pig vas deferens
in which the reticulum is selectively stained with osmium ferrocyanide. Note that the volume of reticulum is less than that seen in the
aorta and that it is predominantly at the periphery of the cells and at the nuclear pole. The reticulum is continuous with the nuclear
envelope and frequently is in close apposition (large arrows) to the mitochondria (m). Nuc indicates nucleus.
firmed previous studies localizing the foot structures (RyR-1)
to the terminal cisternae of the SR in the triad (References 37
and 38 and review in Reference 39) and also established the
efficacy of these antibodies for immunolabeling. We now show
that with the RyR-specific antibodies (anti–RyR 8 –2 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
anti–RyR 8 –1.15 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-mm-thick optical slices of aortic smooth
muscle labeled with either polyclonal anti–RyR 8 –2 or
monoclonal mAb110E antibody, and immunoelectron microscopic analysis of the vas deferens using anti–RyR 8 –1 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 excitationcontraction 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 Ca21 is sequestered in both central and
peripheral SR and that in norepinephrine-stimulated muscles
the Ca21 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 Ca21, but distant from transverse
Lesh et al
183
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Figure 7. Immunoelectron micrographs of guinea pig vas deferens cryosections labeled with anti–RyR 8 –1 and negatively stained with
organotungsten. In panel A, silver-enhanced gold particles (marked with large arrows) can be seen adherent to internal membranes
structures located close to the plasma membrane (marked with arrowheads of portions of two adjacent cells). For comparison, a mitochondrion (M) can be seen in this micrograph. A similar distribution of gold label in the peripheral SR membranes is noted in the micrograph in panel B of portions of two adjacent cells. In some sections, vesicular membranes of the perinuclear SR were labeled, as in
panel C. A large cluster of silver-enhanced gold particles is marked by an arrow, and individual particles associated with membranes
are indicated by arrowheads.
tubules.43 They suggested that these receptors may release Ca21
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 Ca21.48 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 Ca21 pools and the ryanodine-sensitive Ca21
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 micro-
scopic resolution would, however, be necessary to define their
proximity.
Although IP3 is thought to be the physiologically important
mediator of pharmacomechanical Ca21 in smooth muscle
(reviewed in References 1 and 51), vascular and other smooth
muscles also possess a caffeine-sensitive intracellular Ca21 store
that contains sufficient Ca21 to produce, when released,
tension development.45,52 Ca21-induced Ca21 release from
ryanodine receptors, similar to that seen in cardiac53,54 and
skeletal muscle (reviewed in Reference 39), also occurs in various
smooth muscles.7,55–58 Localized domains of high [Ca21]i may exist
near surface couplings of junctional SR59 sufficient to raise local
Ca21 to the '1-mmol/L level required to trigger Ca21-induced
Ca21 release.7,55,60 Caffeine and ryanodine-sensitive Ca21 spikes
originating from the peripheral SR of vascular smooth muscle
184
Ryanodine Receptors in Smooth Muscle
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Figure 8. Distribution of gold particles in immunolabeled guinea
pig vas deferens. Approximately 90% of the silver-enhanced
gold particles were associated with the SR membranes. The
remaining particles were located on nonspecific sites throughout
the cytoplasm and extracellular connective tissue, in the mitochondria, and in the nucleus.
have been observed and are, in many cells, coupled to the
activation of transmembrane cation currents.61 Ca21 sparks contribute to relaxation of cerebrovascular smooth muscle via activation of a plasma membrane Ca21-sensitive K1 channel and plasma
membrane hyperpolarization.62 Activation of Ca21 release
through peripheral RyRs could, then, be gated by dihydropyridine receptors, as in skeletal muscle, or by Ca21, as in cardiac
muscle cells.63 Activation of RyRs in the central SR, however,
would require a diffusible second messenger like Ca21 or cADPribose.64 During excitation-contraction coupling, other factors,
such as phosphorylation65,66 and/or modulators of Ca21 release
from the RyRs, including Mg21, Ca21, pH, calmodulin, and
adenine nucleotides,67 may also alter the physiological responsiveness of these channels in vivo.
Acknowledgments
This study was funded by Public Health Service grant 1K11 AR01871
(Dr Lesh) and National Institutes of Health grants HL-48807(Drs A.P.
and A.V. Somlyo) and HL-32711 (Dr Fleischer). The authors wish to
acknowledge Dr Anthony Timerman for affinity-purifying anti–RyR
8 –1 and anti–RyR 8 –2 and Tarek A. Hijaz and Miles F. Lankford for
providing technical assistance. Figures were prepared with the expert
assistance of Jama Coartney, and we thank Barbara Nordin for her
help in preparing the manuscript.
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Localization of Ryanodine Receptors in Smooth Muscle
Ryan E. Lesh, Graeme F. Nixon, Sidney Fleischer, Judith A. Airey, Andrew P. Somlyo and
Avril V. Somlyo
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Circ Res. 1998;82:175-185
doi: 10.1161/01.RES.82.2.175
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