Role of Sarcoplasmic Reticulum in Arterial Contraction: Comparison ...

have shown that ryanodine is effective on vascular
smooth muscle. 3>2O ~ 22 In contrast to caffeine, which
causes a rapid release of calcium from SR with a
transient contraction, ryanodine usually does not itself
induce a rise in tension. Furthermore, ryanodine does
not appear to affect the contractile apparatus or the
sarcolemmal calcium transport mechanisms. 18 Therefore,
this alkaloid may be especially useful for assessing
the relative role of the SR in vascular smooth muscle
contraction. We compared the effects of ryanodine on
a conduit artery (rat thoracic aorta) and a small
muscular artery (bovine tail artery). This artery is
comparable in size to the rat aorta (about 1.5 mm
diameter) and is particularly interesting because small
resistance vessels (200-300 (xm diameter), whose cells
are virtually identical in morphology to those of the
main tail artery (see "Results"), branch directly from
the tail artery. These functional studies are correlated
with morphological evidence that the rat aorta contains
a much more extensive SR than does the bovine tail
artery. Preliminary reports of some of these findings
have been published in abstract form. 23 - 24
Materials and Methods
Tissues
Small rings of artery (approximately 1.5 mm diameter
and 2-3 mm long) were obtained from bovine tail
artery (distal end) and rat thoracic aorta. Normal
Sprague-Dawley rats (200-300 g) were killed by
decapitation. The thoracic aorta was rapidly excised
and placed into Krebs solution at 37° C. Tails from
normal steers were obtained at a local slaughterhouse.
The distal end of the bovine tail artery was excised
within 1-1 Vi hours of slaughter and placed into Krebs
solution. In all tension experiments, the arteries were
cleaned of surrounding connective tissue and equilibrated
in the warm Krebs solution until resting tension
was stabilized at 500 mg (for at least 1 hour) before data
collection was initiated.
Solutions
The tissues were incubated in a modified Krebs
solution containing (mM) NaCl 138, KC1 4.7,
NaH2PO4 1.2, CaCl2 1.8, MgSO4 1.2, glucose 10,
HEPES 10, adjusted to pH 7.4 with Tris and gased with
100% O2. In some experiments, the potassium concentration
was increased by replacing some of the
sodium chloride with equimolar potassium chloride. In
0-calcium solutions, calcium was replaced with equimolar
MgCl2. In low-sodium solutions (sodium 1.2
mM) Af-methyl-glucamine (138 mM) was used as the
substituting cation and pH was adjusted to 7.4 with
hydrogen chloride.
Special Reagents and Drugs
The following drugs were used: ryanodine (Penick
Corp, Lyndhurst, New Jersey), dantrolene-Na and
diltiazem (Tanabe Co, Japan), nitrendipine (Miles
Pharmaceuticals, Westhaven, Connecticut), verapamil-HCl
(Knoll Pharmaceuticals, Whippany, New
Jersey), /-norepinephrine-HCl and caffeine (Sigma
Ashida et al Ryanodine, Sarcoplasmic Reticulum, and Arterial Contraction 855
Chemical, St. Louis, Missouri), and prazosin-HCl
(Pfizer Laboratories, New York). Dantrolene and
nitrendipine were kept as 10-mM stock solutions in
poryethylenegh/col (#400) in opaque containers.
These agents were added to the Krebs solution as
indicated in "Results."
Contraction Measurements
Two thin (0.4 mm diameter) stainless steel hooks
were inserted through the lumen of the artery ring. One
hook was fixed to the floor of a small jacketed tissue
chamber (volume 0.75 ml); the other hook was
connected to a Harvard Model #52-9529 or Model
#363 force transducer (South Natick, Massachusetts)
that was mounted immediately above the tissue chamber.
Isometric tension was continuously monitored and
recorded on a strip chart recorder. The tissue was
steadily supervised at a rate of 2 ml/min with welloxygenated
incubation fluid at 37° C. Most drugs and
other reagents were added directly to the superfusion
fluids and allowed to reach a steady concentration in the
incubation fluid within the tissue chamber. Usually,
however, norepinephrine (NE) was applied as a small
(25 |il) bolus injection into the superfusion line; the NE
was thus diluted 30-fold when it reached the tissue
chamber. Under these circumstances, the concentration
of NE in the incubation chamber rose rapidly to a peak
that lasted 15-25 seconds; the NE concentration then
declined with a half-time of 15 seconds. Very reproducible
NE-activated contractions were normally obtained
under these conditions (see "Results").
Electron Microscopy
Fresh segments of rat aorta and bovine tail artery (three
animals each) were fixed in 2.5% glutaraldehyde and
postfixed for 30 minutes with 1% OsO4 in cacodylate
buffer. Subsequently, the tissue was dehydrated and
embedded in Epon. Thin sections (60-90 nm) were
poststained with uranyl acetate and lead citrate and
examined in a Zeiss 10 CA electron microscope.
Morphometry
Grids were coded, and random micrographs were
obtained without knowledge of the tissue of origin. If,
at the magnification necessary to visualize SR, the
entire cell cross-section could not be included, a
random region of the cell that included the cell surface
and part of the nucleus was chosen. Electron micrographs
of four to six arterial smooth muscle cells from
each animal (three rats and three steers) were taken on
a random basis and enlarged to a final magnification of
25,000 x. The micrographs were evaluated morphometricalry
using the point grid planimetry method. 25 A
1-cm point grid was used for quantification of nucleusfree
cell volume. The area of SR (membrane-bound
cytoplasmiccisternae, tubules, and vesicles) and rough
endoplasmic reticulum (RER; ribosome-covered membranes)
was quantitated with a 0.5-cm point grid. These
data were expressed as volume percent of nucleus-free
cell volume, giving a proportional estimate of SR and
RER content in the cytoplasm.
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