10%
/J-Adrenergic Receptor Distribution
Among Muscle Fiber Types and
Resistance Arterioles of White, Red, and
Intermediate Skeletal Muscle
Wade H. Martin III, Sidney S. Murphree, and Jeffrey E. Saffitz
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The pathophysiological effects of congestive heart failure and physiological effects of exercise
training on skeletal muscle may be mediated in part by modulation of 0-adrenergic receptor
density. To shed light on the physiological role of skeletal muscle ^-receptors, their density and
distribution were characterized in muscle fibers and resistance arterioles of whole tissue slices
of three rat hindquarter muscles differing markedly in fiber type composition and capacities for
oxidative metabolism and vasodilatation. Binding isotherms and quantitative light microscopic
autoradiographic localization of receptors were performed by incubating tissue slices in selected
concentrations of [l2SI]cyanopindolol with and without 10"' M /-propranolol. Muscle fiber types
were delineated hi adjacent sections by histochemical staining of myofibrillar ATPase activity at
pH 4.5-4.55. The total tissue content of receptors (Bn^J was nearly threefold greater in the
soleus, a muscle consisting almost entirely of slow-twitch (type I) fibers than in superficial white
vastus lateralis, a muscle composed of >95% fast-twitch (type lib) fibers. Bnax was intermediate
in gastrocnemius, a mixed fiber muscle (all differences p<0.01). Receptor affinity for radioligand was higher in the white vastus than in the mixed fiber and slow-twitch muscles (Kt=
3.5±0.4 pM for white vastus versus 6.8±0.8 and 6.4±1.1 pM in gastrocnemius and soleus,
respectively; both y?<0.01 versus white vastus). Disparities in B ^ among muscles were due
entirely to differences hi receptor densities of skeletal muscle cells as shown autoradiographically. Furthermore, variations in B ^ of the three skeletal muscles were directly related to
percentage of type I fibers (r=0.99; p<0.001), which had a /3-receptor density that was
approximately 4.5-fold greater than hi superficially located type lib fibers, 3.2-fold greater than
in intermediate depth type lib fibers, and 2.0-fold greater than in type Ila fibers. In contrast,
gram densities of resistance arterioles were similar regardless of surrounding skeletal muscle
fiber type composition. However, resistance arterioles were 2.5- and 6.1-fold more numerous in
the slow-twitch soleus than in the gastrocnemius and superficial white vastus, respectively (all
differences /?<0.01). We conclude that /3-receptor density of rat hindquarter skeletal muscles is
directly proportional to percentage of slow-twitch fibers, while receptor affinity for antagonist
is higher in fast-twitch than in slow-twitch or mixed fiber muscles, ^-receptor density of
resistance arterioles is similar among types of muscles but these vessels are far more numerous
in the slow-twitch soleus. Such differences reflect the diverse metabolic and physiological
characteristics of the rat hindquarter musculature. {Circulation Research 1989;64:1096-1105)
R
educed exercise tolerance in patients with
chronic congestive heart failure may be
due in part to skeletal muscle abnormalities
From the Departments of Medicine and Pathology, Washington University School of Medicine, St. Louis, Missouri.
Supported by National Institutes of Health NHLBI Grant
HL-17646, SCOR in Ischemic Heart Disease. J.E.S. is an
Established Investigator of the American Heart Association.
Address for reprints: Jeffrey E. Saffitz, MD, PhD, Department
of Pathology, Washington University School of Medicine, Box
8118, 660 S. Euclid Ave., St. Louis, MO 63110.
Received July I, 1988; accepted November 4, 1988.
that include impaired capacity for vasodilatation
and decreased reliance on aerobic metabolism. '~3 In
contrast, endurance exercise training enhances both
of these capacities.4-6 Mechanisms responsible for
these effects are not understood. However, fat and
carbohydrate metabolism and microvascular tone
are influenced markedly by /3-adrenergic activity.7-8
Recent studies indicate that /3-adrenergic receptor
density is decreased in the myocardium of patients
with congestive heart failure in conjunction with a
reduction in contractile response to sympathetic
Martin et al
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agonists.9 Moreover, /3-receptor density is decreased
in skeletal muscle of dogs with pacing-induced heart
failure10 and increased in the exercise-trained musculature of rats." Thus, pathophysiological alterations in adrenergic responsiveness of skeletal and
cardiac muscle may be mediated by regulation of
/3-adrenergic receptor density.
Various skeletal muscles of the rat hindquarter
differ markedly in velocities of contraction and
capacities for vasodilatation and aerobic metabolism.12-15 These properties are related closely to
muscle fiber type population. l2~is Muscles such as
the superficial white vastus lateralis, composed
almost entirely of fast-twitch glycolytic fibers, have
low rates of aerobic metabolism and blood flow
during exercise while muscles such as the soleus
and gastrocnemius, composed primarily of slowtwitch and fast-twitch oxidative glycolytic fibers,
respectively, have severalfold higher rates of oxidative metabolism and perfusion under the same
conditions. Results of a recent investigation indicate that /3-receptor density differs among muscles
with disparate oxidative capacities." However, in
this and most previous studies, /3-receptor density
was assessed in crude membranes prepared from
tissue homogenates. Thus, the distribution of /3receptors among muscle fibers and vascular components of muscles having different vasodilatory and
oxidative capacities is unknown.
In this investigation, we used quantitative light
microscopic autoradiography to delineate the distribution of skeletal muscle fiber and resistance
vessel /3-receptors and characterize differences in
/3-receptor density among fiber types and subtypes
of three skeletal muscles of the rat hindquarter
having dissimilar capacities for aerobic metabolism and vasodilatation.
Materials and Methods
Preparation of Tissue Slices
The gastrocnemius, soleus, and vastus lateralis
muscles were isolated by blunt dissection and excised
from 9-10-week-old sedentary Sprague-Dawley rats
that had been anesthetized with intraperitoneaJ pentobarbital. Muscles were frozen immediately in liquid nitrogen (from which dissolved air had been
withdrawn under a vacuum) and stored at -70° C in
sealed containers until used in experiments.16 Unfixed
frozen sections of skeletal muscle, 12 fim in thickness, were cut in cross section with a cryostat
microtome. Sections were placed on gelatin-coated
glass slides for radioligand binding and light microscopic autoradiographic studies. Alternate serial sections were mounted on coverslips for histochemicaJ
delineation of myofibrillar ATPase or NADHtetrazolium reductase activities.
Radioligand Binding Assays
Unfixed, slide-mounted frozen sections of skeletal muscle were incubated for 60-180 minutes at
Distribution of Skeletal Muscle /3-Receptors
1097
37° C in buffer (NaCl 154 mM, MgCl2 10 mM,
Tris-HCl 10 mM, pH 7.4) containing selected
concentrations of the /3-adrenergic antagonist
(-)-[125iodo]cyanopindolol (ICYP) (2,200 Ci/mmol,
New England Nuclear, Inc, Boston, Massachusetts) in the presence or absence of unlabeled
/-propranolol. Nonspecific binding was defined as
binding of radioligand in the presence of 10~5 M
/-propranolol, which was kindly provided by Ayerst
Laboratories, New York, New York. Sections were
incubated with radioligand in large volumes of buffer
(40-50 ml) so that the concentration of free radioligand did not change measurably during the incubations. Nonspecifically bound radioactivity was
removed by incubating slides for 1 hour in buffer at
22° C without radioligand or unlabeled displacer.
After rinsing, sections were dipped briefly in distilled water to remove buffer solutes, dried under a
gentle stream of air, and either scraped off slides for
quantification of radioactivity by gamma scintillation spectrometry or prepared for autoradiographic
analysis as described below. In preliminary experiments, radioligand binding conditions, association
kinetics, and rinsing conditions for selective removal
of nonspecifically bound radioactivity were characterized in rat skeletal muscle (data not shown) and
found to be virtually identical to conditions reported
previously for optimal assessment of radioligand
binding to unfixed frozen sections of canine
myocardium.17
Radioactivity of whole tissue slices was determined by scraping sections from slides with a razor
blade and quantifying radioactivity in individual
sections by gamma scintillation spectrometry (85%
counting efficiency). Radioactivity was expressed
per milligram protein measured in selected sections
with the Lowry protein assay18 or per square centimeter tissue cross-sectional area determined by
digitization of carefully traced outlines of enlarged
photographs of selected sections. Protein content
per square centimeter cross-sectional area was equivalent in the three muscles analyzed.
Quantitative Autoradiography
Radioligand binding sites were localized autoradiographically in skeletal muscle sections using the
emulsion-coated coverslip method employed in previous studies of cardiac muscle /3-receptors.17 Acidwashed, gelatin-coated coverslips were coated with
Kodak NTB2 nuclear track emulsion (Eastman
Kodak Co, Rochester, New York) and dried at
room temperature for at least 3 hours. The emulsioncoated coverslips were glued at one end to slides
containing radiolabeled sections, and, after exposure of the emulsion to underlying radioactivity for
18-30 hours, unglued edges of each coverslip were
gently lifted from slides, and emulsions were developed for 4 minutes with Kodak D19 developer diluted
1:1 with water and fixed for 4 minutes at 25° C with
Kodak fixer. After photographic processing, tissue
sections were stained with hematoxylin-eosin, and
1098
Circulation Research Vol 64, No 6, June 1989
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coverslips were sealed permanently to the slides.
The tissue and overlying developed grains in the
emulsion layer were examined by light microscopy.
Multiple grain density measurements were made in
discrete regions composed of distinct populations of
skeletal muscle fibers or resistance arterioles (10100 fitn in diameter). Because adjacent serial sections were used for histochemical staining of myofibrillar ATPase and NADH-tetrazolium reductase
activities and autoradiography, it was possible to
locate identical cells in serial tissue cross sections.
Thus, grain densities were quantified in groups of
skeletal muscle fibers corresponding to the same
cells observed in histochemically stained sections.
Grain densities were measured with a computerassisted planimeter interfaced by video camera to a
light microscope. Test regions delineated by a box on
the video monitor and encompassing 3,312 fim2 of
section area were randomly selected in desired groups
of fibers identified by histochemistry. The number of
grains was determined within each test region and
expressed as grains per 104 /xm2. Specific grain densities were calculated by subtracting grain densities
in test regions of sections incubated with radioligand
plus 10"3 M propranolol (grains attributable to nonspecific binding of radioligand plus photographic
background) from measurements made in corresponding test regions of sections incubated with radioligand only (total binding). At least 20 random grain
density measurements were made in each of multiple
regions in at least three separate tissue sections.
Histochemical Studies
Muscle fiber type populations and capacities of
individual fibers for oxidative metabolism were delineated by histochemical staining of myofibrillar
ATPase and NADH-tetrazolium reductase activities, respectively. The former method involved preincubation of tissue sections attached to coverslips
in buffer at pH 4.5-4.55, followed by incubation in
buffer containing 3.6 mM ATP and 18 mM calcium
chloride, and subsequent sequential incubations
with 1% calcium chloride, 2% cobalt chloride, and
1% ammonium sulfide.19 Under these conditions
slow-twitch fibers exhibit dark staining features and
fast-twitch fibers may be differentiated into light
staining type Ua and intermediate staining type lib
subtypes.20 To further categorize type II fibers on
the basis of oxidative capacity, sections were stained
for NADH-tetrazolium reductase activity with the
procedure of Novikoff.21
Quantification of Resistance Arterioles
The number of arterioles in selected regions of
the three hindquarter muscles was determined in
paraffin-embedded sections of formalin-fixed tissue.
Muscles were dissected from rat hindquarters as
described and fixed by immersion in 10% buffered
formalin. Tissue was processed conventionally for
light microscopy, embedded in paraffin, and crosssectioned at a thickness of 5 ^.m. Hematoxylin-
eosin and Verhoff-van Gieson stained sections were
examined by light microscopy, and the number of
sectional profiles of resistance arterioles (10-100
fim in diameter) was counted in selected regions.
Data were expressed as number of arteriolar profiles per square millimeter tissue area measured
with computer-assisted planimetry.
Statistical Analysis
Data are expressed as mean±SD. Binding isotherm data were transformed according to the
method of Scatchard.22 Simple linear regression
was used in determining intercepts and slopes in
Scatchard plots. The statistical significance of differences in Bnua, KA, grain density measurements,
and vessel number measurements was determined
within and among muscles from the same animals
by one-way analysis of variance. Least squares
linear regression analysis was used to assess the
correlation between percentage of type I myocytes
and grain density measurements or resistance vessel number in various regions. Statistical differences were considered significant at p<0.05.
Results
Radioligand Binding
The total tissue content of/3-receptors in soleus,
gastrocnemius, and superficial white vastus lateralis
skeletal muscles was determined with binding isotherms in whole tissue sections from four different
animals. As shown in Figure 1, binding of radioligand was rapid, saturable, and of high affinity.
Specific binding was greater than 90% of total
binding at near saturating radioligand concentrations.
Scatchard plots were linear for all three hindquarter
muscles. In the examples shown in Figure 1, B,™, was
fourfold greater in soleus than in superficial white
vastus lateralis muscle and intermediate in gastrocnemius. Mean data, shown in Table 1, indicated that
/3-receptor density in soleus was 1.8 times greater
than in gastrocnemius and 2.9 times greater than in
white vastus (all differencesp<0.01). Receptor affinity
for radioligand (Aj) was nearly twofold greater in
white vastus than in either soleus or gastrocnemius
(both p<0.01 versus white vastus; Table 1).
Light Microscopic Autoradiography
The distribution of/3-adrenergic receptors in muscle fiber and resistance vessel components of each
hindquarter skeletal muscle was delineated by light
microscopic autoradiography. In initial experiments, grains were counted in randomly selected
test areas of muscle fibers throughout cross sections
without reference to histochemically distinct regions.
As shown in Figure 2, differences in mean B ^
values of the three hindquarter muscles were
accounted for entirely by differences in mean grain
densities of fibers of each muscle. Thus, ratios of
Brau (determined with binding isotherms) and of
myocytic grain density values of white vastus to
Martin et al Distribution of Skeletal Muscle ^-Receptors
SOLEUS
4
a.
U
4000
1099
28lmol/mg protein
K d . 7pM
2000
5
60
120
180
[FREE ['251]CYP](pM)
Q-
5
10 15 20 25 30
BOUND (fmol/mg protein)
240
GASTROCNEMIUS
2.0
15000
10
?L 5000
s
20
40
60 80 100
[FREE [ 1 2 5 I]CYP] (pM)
a.
U
11 fmol /mg pfolam
7pM
120
2 4 6 8 10 12 14
BOUND(fmol/mg protein)
WHITE VASTUS LATERALIS
2000
FIGURE 1. Binding isotherms and Scatchard plots of (—)-{miodo]cyanopindolol
(ICYP) binding to whole tissue slices of rat
soleus, gastrocnemius, and white vastus
lateralis skeletal muscles. Sections were
incubated under equilibrium binding conditions at37"C with ICYP (0.9-220 pM),
rinsed, dried, scraped, and radioactivity
was quantified by gamma scintillation spectrometry. Isotherm plots are means of
triplicate determinations of total (•), nonspecific (0), and specific (•) binding. CPM,
counts/min; BIF, bound/free.
1000
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5
a.
20
40
[FREE['
60
25
80
100
2
I]CYP]( P M)
4
6
8
10
BOUND (fmol/mg protein)
soleus were nearly identical (0.34 and 0.35, respectively). Corresponding ratios of gastrocnemius to
soleus were also very similar (0.56 for B ^ and 0.51
for grain density; NS).
In additional experiments performed to characterize relations between /3-receptor density and
fiber type population within and among hindquarter
muscles, grain densities were quantified over
selected areas of each muscle that contained varying proportions of type I and type II fibers. Examples of these regions are shown in Figure 3. Analyses were performed in three regions of soleus (S-l,
S-2, and S-3), six regions of gastrocnemius (G-l
through G-6), and two regions of white vastus
(WV-1 and WV-2). Areas selected for these autoradiographic analyses were composed of the following percentages of type I fibers: 100% (S-l), 74%
(S-2), 54% (G-l), 40% (S-3), 37% (G-2), 24% (G-3),
19% (G-4), 15% (G-5), 0% (G-6), and 0% (WV-1
and WV-2). Grain densities of all regions were
normalized to values observed in areas of soleus
containing 100% type I fibers. There was a linear
increase in grain density of regions containing progressively greater proportions of type I muscle
fibers (Figure 4) up to the maximal value of 1.0 for
regions of soleus consisting entirely of type I fibers.
Regions of white vastus and gastrocnemius containing no type I fibers had mean normalized grain
densities of 0.22, 0.23, and 0.24. Selected microscopic regions of soleus containing 40% and multiple large areas of gastrocnemius containing 37%
type I fibers had similar normalized grain densities
(0.55 and 0.58; NS). Differences in normalized grain
density among regions of gastrocnemius containing
54%, 37%, 24%, 15%, and 0% type I fibers and
300
•
Q
•
SCX.EUS
GASTROCNEMIUS
WHITE VASTUS
200
IS)
<
at
100
O
TABLE 1. Total Tissue Content ( B ^ and Affinity (KJ of /JAdrenergic Receptors in Three Rat Hindqnarter Skeletal Mosdes
Muscle
Soleus
(n=4)
Gastrocnemius
(«=4)
Superficial white
vastus lateralis
(n=4)
(fmol/cm2 area) * d (pM)
23.5±5.4
6.4±l.l
13.1±2.4*
8.l±0.7*t
Data are mean±SD
•p<0.01 vs. soleus.
tp<O.OI vs. gastrocnemius.
MYOCYTES ARTERKXES
B
GRAIN DENSITY
1.0
6.8±0.8
0.56
3.5±0.4*t
0.34
FIGURE 2. Mean B-adrenergic receptor density of muscle fiber and resistance arteriolar components of rat
soleus, gastrocnemius, and white vastus lateralis skeletal
muscles determined by light microscopic autoradiography of whole tissue slices. Mean B-receptor density,
expressed as specific grains per 104 fim1 tissue area,
reflects specific binding of (—)-{l2Siodo]cyanopindolol to
musclefibersor resistance arterioles. *p<0.0I vs. soleus;
tp<O.OI vs. gastrocnemius.
1100
Circulation Research Vol 64, No 6, June 1989
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4
FIGURE 3. Examples of selected areas offrozen sections of rat soleus (upper panels), gastrocnemius (center panels),
and white vastus (lower panels) showing relation between proportion of type I fibers and ^-receptor density. Adjacent
serial sections were stained for myofibrillar ATPase activity at pH 4.5—4.55 (left component of each panel) or incubated
with radioligand and prepared for autoradiography (right component of each panel). The region of soleus shown consists
entirely of dark staining type I fibers having a high receptor density. The region of white vastus shown consists entirely
of type lib fibers and has a low receptor density. The region of gastrocnemius shown contains approximately 25% type
I fibers and 75% type lib fibers and has an intermediate receptor density. Detailed grain density analyses were performed
in selected microscopic fields of soleus containing 40-100% type I fibers, areas of gastrocnemius containing 0-54% type
I fibers, and areas of white vastus containing 0% type I fibers as described in the text. Bar, 100 fim.
among regions of soleus containing 100%, 74%, and
40% type I fibers were all significant (p<0.01).
Thus, ^-adrenergic receptor density among and
within hindquarter muscles is directly proportional
to the percentage of type I fibers, regardless of their
anatomic location.
To further evaluate the effects of anatomic location (superficial versus intermediate depth), myo-
cyte subtype (Ha versus lib), and oxidative capacity of muscle fibers on ^-receptor density, additional
studies were conducted on entire vastus lateralis
muscles. Selected regions of adjacent serial sections stained for myofibrillar ATPase and NADHtetrazolium reductase activities and studied autoradiographically are shown in Figure 5 and illustrate
the progressive transformation from a predom-
Martin et al Distribution of Skeletal Muscle 0-Receptors
1-0 r
s-i •
r • 0.99 (p< 0.001)
y0.0075X • 0.2225
25
50
75
100
%TYPE 1 FIBERS
(Myofibrillar ATPase pH 4.5-4.55)
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FIGURE 4. Relation between proportion of type I fibers,
delineated by staining of myofibrillar ATPase activity at pH
4.5—455, andfi-adrenergicreceptor density determined by
light microscopic autoradiography. Specific grain densities
were measured in three areas of sole us (S-l through S-3)
containing 40-100% type I fibers, six areas of gastrocnemius (G-l through G-6) containing 0-54% type Ifibers,and
two areas of white vastus (WV-1 and WV-2) containing 0%
type I fibers. Points are means of at least 20 random grain
density measurements in each region.
inance of fibers having intermediate intensity staining characteristics for ATPase (type lib) and a low
oxidative capacity (light staining NADH) at the
periphery to a predominance of fibers having either
light or dark staining ATPase characteristics (types
Ila and I, respectively) and a higher oxidative
capacity (darker staining NADH) at intermediate
and deep levels. Grain densities were measured in
selected groups of fibers of vastus muscles located
at superficial (region 1) and intermediate (region 2)
depths and consisting of 100% type lib fibers and in
clusters of cells from deep regions consisting of
100% type Ila (region 3) and 100% type I (region 4)
fibers. Normalized grain densities of each region
were 0.22 for superficial type lib fibers, 0.31 for
intermediate depth type lib fibers, 0.49 for type Ila
fibers, and 1.0 for type I fibers (all differences
p<0.05). Thus, relative grain densities for type I and
lib fibers of the vastus are similar to values previously observed for type I and II fibers in the gastrocnemius and soleus. However, the grain density of
highly oxidative type Ila muscle fibers (as delineated
by NADH-tetrazolium reductase activity) is intermediate between that of type I and lib fibers.
Resistance Vessels
In contrast to results for skeletal muscle fibers,
there were no significant differences in /3-receptor
densities of arterioles of the three hindquarter skeletal
muscles. As shown in Figure 2, mean grain densities
of resistance vessels ranging in diameter from 10 to
1101
100 fim were equivalent for the soleus, gastrocnemius, and white vastus. Despite the homogeneity of
/3-receptor density among these vascular components, large differences were observed among hindquarter muscles in the mean number of resistance
vessels per square millimeter cross-sectional tissue
area (Table 2). The mean number of sectional profiles
of resistance vessels varied sixfold among muscles
from 1.0±0.2 vessels/mm2 tissue area for superficial
white vastus lateralis to 6.0± 1.1 vessels/mm2 area for
soleus. Resistance vessel number in the gastrocnemius was intermediate. Differences among all three
muscles were highly significant (all /?<0.01).
Regional differences in resistance vessel number
within muscles were assessed in three areas of the
gastrocnemius and vastus lateralis that contained
markedly divergent proportions of type I and II
fibers. In the gastrocnemius, there was a significant
difference (44%; p<0.05) in vessel number between
large superficial areas containing virtually no type I
fibers and small deep areas consisting of approximately 40% type I fibers. However, in the vastus
lateralis, vessel number increased markedly (451%)
and progressively from superficial to deep regions
in conjunction with a change in fiber type composition from 0% type I in superficial portions to >70%
type I in the deepest areas of muscle immediately
adjacent to the underlying bone (Figure 5; Table 2).
In the deep areas of the vastus, the number of
vessels per square millimeter cross-sectional tissue
area and the proportion of type I fibers were nearly
equivalent to corresponding values in the soleus.
There was a highly significant correlation (r=0.96;
p<0.001) between percentage of type I fibers and
number of resistance vessels in the three hindquarter muscles. Thus, although the /3-adrenergic receptor density of individual resistance arterioles is
uniform among and within rat hindquarter skeletal
muscles, the number of resistance vessels appears
to be closely related to the proportion of type I
myocytes in the surrounding muscle tissue.
Discussion
The purpose of this investigation was to characterize the density and distribution of /J-adrenergic
receptors in fibers and vascular components of rat
hindquarter skeletal muscle. /3-Receptors regulate a
variety of physiological processes such as fat and
carbohydrate metabolism, vasodilatation, and the
contractile state of cardiac and skeletal muscle. 7 - 912
Muscles of the rat hindquarter provide an excellent
experimental system for investigation of functional
roles of ^-receptors because they differ markedly in
mechanical properties and capacities for vasodilatation and oxidative metabolism. We observed a
1.8-fold to threefold greater /3-receptor density in
the slow-twitch soleus versus the gastrocnemius
and superficial white vastus lateralis muscles, respectively, which contain much greater proportions of
fast-twitch fibers. Earlier studies had reported conflicting results regarding relative /3-receptor densi-
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FIGURE 5. Areas selected from the deep (upper panels), intermediate (center panels), and superficial (lower panels) portions of rat vastus lateralis skeletal muscle.
Adjacent serial sections were stained for myofibrillar ATPase activity at pH 4.5-4.55 {left component of each panel), NADH-tetrazolium reductase activity {center
component of each panel) or incubated with radioligand and prepared for autoradiography (right component of each panel). Lowercase letters delineate the locations
of identical cells in the ATPase {right panels) and NADH-tetrazolium reductase {center panels) stained sections. Autoradiographs are shown at considerably higher
magnification in order to illustrate relative grain densities of regions composed exclusively of type I fibers {right upper panel), type Ha fibers (right center panel), and
type lib fibers (right lower panel). Deep portions consist largely of type I fibers (dark ATPase staining) with high oxidative capacity (intense staining for
NADH-tetrazolium reductase activity) and an abundance of ^-receptors relative to superficial regions composed of type lib fibers (intermediate ATPase staining
intensity) having a low oxidative capacity and ^-receptor density. Intermediate zones are composed of a mixture of type I, type Ha (light ATPase staining), and type
lib fibers having variable oxidative capacities and intermediate ^-receptor density. Grain densities are lower than in examples shown in Figure 3 due to a shorter
emulsion exposure interval. Detailed grain density analyses were performed in clusters of fibers of vastus lateralis as described in the text. Bar, 300
D
o
n
Martin
TABLE 2. Number of Resistance Arterioles and Proportion of
Type I Muscle Fibers Within Various Regions of Three Rat
Hindquarter Skeletal Muscles
Muscle
Soleus (n=5)
Gastrocnemius
(«=5)
Region
Entire muscle
Superficial
Intermediate
Deep
Vastus lateralis Superficial
(n=5)
Intermediate
Deep
Resistance
arterioles
(number/mm2)
6.0±l.l
2.6±0.7*
2.9±0.9*
3.7±0.5*tt
1.0±0.2*§
1.9±0.6*t§
5.5±1.5t«
%Typel
Fibers
84.4+5.1
0*
19.5±4.4*t
43.9±4.5*tt
0*
13.0±2.8*t§
71.3±13.1tt§
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Data are mean±SD. The relation between the proportion of
type I fibers (x) and the density of resistance arterioles (y) in
each region was characterized with linear regression analysis
(y=0.052j+1.65; r=0.96, p<0.001).
*p<0.01 vs. soleus.
tp<0.02 vs. superficial region of same muscle.
tp<0.02 vs. intermediate region of same muscle.
§p<0.05 vs. same region of gastrocnemius.
ties of slow- and fast-twitch muscles. Reddy et al23
reported fewer ^-receptors in whole tissue slices of
slow-twitch rat skeletal muscle while WatsonWright and Wilkinson24 found a twofold greater
density of /3-receptors in tissue slices of slowversus fast-twitch muscles of mice. Williams et al"
demonstrated that membranes prepared from tissue
homogenates of the soleus contained 2.5-3 times as
many /3-receptors as those from the predominately
fast-twitch gastrocnemius. Our studies of whole
skeletal muscle tissue slices are in general agreement with the findings of Watson-Wright and
Wilkinson24 and Williams et al." Furthermore, we
have extended this concept to the superficial white
vastus lateralis, which contains an even lower density of /3-receptors and slow-twitch myocytes than
the gastrocnemius and has provided definitive evidence that total /3-receptor density of skeletal muscle is closely related to fiber type population. Williams et al" reported a correlation coefficient of 0.63
between oxidative capacity, defined by succinate
dehydrogenase activity, and /3-adrenergic receptor
density of gastrocnemius muscle. Although we did
not measure oxidative capacity, we demonstrated
the existance of a close relation (r=0.99) between
percentage of type I fibers and /3-receptor density in
three hindquarter skeletal muscles with diverse
fiber type populations. Oxidative capacity of type I
myocytes is approximately threefold higher than
that in type lib myocytes but less than half that of
type Ha fibers.23 Armstrong and Phelps20 found that
gastrocnemius muscle, particularly its large superficial and middle portions, is primarily composed of
type lib fibers that have a low oxidative capacity
while soleus consists almost exclusively of type I
fibers that contain higher concentrations of oxidative enzymes. Our results suggest that the significant relation between oxidative capacity and /3receptor density reported by Williams et al" largely
et al
Distribution of Skeletal Muscle ^-Receptors
1103
reflects differences in fiber type population. Further
support for this interpretation is provided by our
autoradiographic data demonstrating that /3receptor density is nearly identical for myocytic
components of regions of different hindquarter muscles, including the gastrocnemius and soleus, that
contain similar percentages of type I and type II
fibers.
Results of the present studies indicate that receptor affinity for radioligand is approximately 80%
greater in fast-twitch vastus lateralis muscle than in
the soleus. These data are in general agreement with
previous results of Watson-Wright and Wilkinson,24
who noted a higher affinity for antagonist in fastversus slow-twitch skeletal muscle. Williams et al"
found that affinity for antagonist was lower in the
soleus than in the mixed fiber gastrocnemius muscle
whereas we did not observe a difference in radioligand binding affinity between these two muscles.
The reason for this discrepancy is unclear but may
be related to dissimilarities in radioligand binding to
whole tissue slices and membranes prepared from
tissue homogenates. Receptor affinity was more
than one order of magnitude higher in the tissue
slice preparation.
The physiological importance of variations in
/3-receptor density and/or affinity among myocytic
components of skeletal muscle is not known. Fundamental differences exist in physiological effects of
/3-agonist stimulation on mechanical properties and
metabolism of fast- and slow-twitch skeletal muscle. Bowman and Zaimis12 first described the disparate influence of isoproterenol on the rate and
extent of tension development in these two types of
muscle. The magnitude of these effects was approximately twice as large in feline soleus as in the
fast-twitch anterior tibialis, possibly reflecting differences in /3-receptor density such as we have
observed. In addition, epinephrine substantially augments glycogen breakdown in slow-twitch muscle
during electrical stimulation but has no additive
effect beyond that of contractions alone in fasttwitch muscle.26 The greater density of/3-receptors
in slow-twitch myocytes may enhance carbohydrate substrate availability in these fibers during
intense muscular work when catecholamine levels
are likely to be elevated and the usual supply of
substrates used by slow-twitch fibers for aerobic
metabolism at rest and during mild exercise is
insufficient. Other investigations have indicated that
^-blockade inhibits intramuscular triglyceride use
during prolonged exercise in slow- but not fasttwitch fibers, which do not oxidize a significant
amount of fat.27 Further studies are needed to
delineate the functional importance of variations in
/3-receptor density and affinity among different fibers
of slow- and fast-twitch muscles.
Our results indicate that /3-receptor densities of
resistance arterioles of the three muscles studied
were equivalent. The vascular /3-receptor density
was greater than that of type II fibers but less than
1104
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Vol 64, No 6, June 1989
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that of type I fibers. Lavenstein et al28 reported a
much higher density of /3-receptors in vascular
versus muscle fiber components of rat hindquarter
muscle. However, in these studies, radioligand was
administered by vascular perfusion which likely
accounts for the 10-100-fold greater binding
observed on vascular components. Other investigations of/3-receptors in skeletal muscle have involved
either membranes prepared from tissue homogenates or whole tissue slices that were not analyzed
autoradiographically, thus precluding localization
of receptors on specific cellular components. Laughlin and Armstrong29 showed that propranolol reduced
blood flow to a similar extent in fast- and slowtwitch muscles of the rat hindquarter at rest and
during mild exercise. Gray30 found that sensitivity
to vasoconstriction induced by norepinephrine was
greater in fast- than in slow-twitch rat skeletal
muscle but did not detect a difference in vasodilatory responses to epinephrine. These observations
are consistent with our data demonstrating no difference in /3-receptor density among resistance vessel components of skeletal muscles containing divergent populations of type I and type II fibers.
Resistance arterioles 10-100 ^.m in diameter were
2.4-6.1-fold more numerous in the slow-twitch
soleus and in the deep red vastus than in the
gastrocnemius and superficial white vastus muscles, both of which consist predominately of fasttwitch fibers. Although a direct relation between the
proportion of slow-twitch fibers and vascularity was
found (r=0.96), we were surprised that differences
in vascularity among regions of skeletal muscle
reflected the surrounding myocyte population to a
greater extent in the vastus lateralis than in the
gastrocnemius. Small deep areas of the gastrocnemius contained a significant proportion of type Ila
fibers, which have a fourfold higher capacity for
aerobic metabolism than type lib fibers, which
account for more than 95% of the superficial portions of both the gastrocnemius and vastus. 20 ^
Thus, muscle blood flow during electrical stimulation or exercise may be coupled to oxidative capacity as well as fiber type but varies markedly between
the deep red and superficial white portions of both
muscles. 1431 Previous studies had reported conflicting findings regarding the correlation between
capillary density and oxidative capacity of mammalian skeletal muscle32-34 and suggested that differences in blood flow among muscles were related to
other anatomic factors such as variations in number
and size of resistance vessels or differences in
responses of the vasculature to humoral or metabolic stimuli. Our data demonstrate that resistance
vessel number is generally greater in muscle tissue
consisting predominately of slow-twitch and fasttwitch oxidative glycolytic fibers but that the relation between resistance vessel number and oxidative capacity or fiber type is complex and reflects
not only physiological but anatomic factors such as
tissue depth and location. Although the functional
basis for this effect is unclear, the gastrocnemius,
plantaris, and soleus may function as a physiological unit resembling the entire vastus lateralis with
regard to localization of fast-twitch fibers for use in
"fight or flight" responses at the periphery and
placement of slow-twitch fibers adjacent to the bone
for maintenance of posture.
In summary, we have shown that /3-receptor
density of myocytic components varies markedly
within and among skeletal muscles of the rat hindquarter in direct proportion to percentage of type I
fibers. In contrast, /3-receptor affinity of muscle
fibers was greater in fast than in slow-twitch muscle. Density of/3-receptors was similar among resistance vessels of these hindquarter muscles, but
these vessels were severalfold more numerous in
the slow-twitch soleus. Further studies are needed
to clarify the metabolic and physiological implications of these findings.
Acknowledgments
We thank Timothy K. Tolley for technical assistance; John O. Holloszy, MD, for providing rat
skeletal muscles; and Susan Johnson for preparation of the typescript.
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KEY WORDS • ^-receptors • autoradiography
muscle
• skeletal
Beta-adrenergic receptor distribution among muscle fiber types and resistance arterioles of
white, red, and intermediate skeletal muscle.
W H Martin, 3rd, S S Murphree and J E Saffitz
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Circ Res. 1989;64:1096-1105
doi: 10.1161/01.RES.64.6.1096
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