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Localization of calcium in vas deferens using 45Ca EM autoradiographyRelationship to species and the effect of 45Ca removal.

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THE ANATOMICAL RECORD 217:321-327 (1987)
Localization of Calcium in Vas Deferens Using
45Ca EM Autoradiography: Relationship to Species
and the Effect of 45CaRemoval
LINDA J. McGUFFEE, SALLY A. LITTLE, AND BETTY J. SKIPPER
Department of Pharmacology (L.J.
M., S . A . L ) and Department of Family, Community,
and Emergency Medicine (B.J.S.), School of Medicine, University of New Mexico,
Albuquerque, NM 87131
ABSTRACT
The distribution of calcium was determined in the vas deferens of
the guinea pig using 45Ca electron microscopic autoradiography of rapidly frozen,
freeze-dried, and embedded tissue. A selective accumulation of calcium at the plasma
membrane and SR was observed in vas deferens that had been incubated in 45Ca for
65-85 min prior to rapid freezing. Rinsing the tissue in nonradioactive calcium for
6 min prior to rapid freezing significantly altered the distribution of calcium among
the plasma membrane, mitochondria, and cytoplasmic matrix. The influence of
species on the observed distribution of calcium was also examined. The distribution
of calcium in the guinea pig vas deferens was not significantly different from that
in the rabbit vas deferens when the tissues were prepared under identical conditions.
The role of calcium in smooth muscle contraction has
been studied extensively, and both extracellular and
intracellular pools of activator calcium have been identified (Daniel et al., 1983; Hudgins and Weiss, 1968;
Johansson and Somlyo, 1980; Van Breemen et al., 1980).
However, the ultrastructural locations of the extracellular or intracellular activator calcium have not been
determined. It has been postulated that the clear, membrane-bound spaces (presumably sarcoplasmic reticulum), mitochondria, and plasma membrane may serve
as sites of intracellular calcium sequestration (Daniel et
al., 1983; Johansson and Somlyo, 1980; McGuffee et al.,
1981; Popescu and Diculescu, 1975; Sugi and Daimon, 1977; Suzuki, 1982), although the relative contribution of these sites as sources of activator calcium has
only recently begun to be investigated using morphological techniques (Bond et al., 1984a; McGuffee et al.,
1985).
Our laboratory has been examining the usefulness of
45Ca autoradiography (ARG) at the electron microscopic
(EM) level of resolution in evaluating calcium distributions in smooth muscle. Two requirements must be met
before one can examine the distribution of a soluble ion
such as calcium. First, tissue ultrastructure must be
sufficiently preserved so that intracellular organelles
can be identified. Second, the distribution of calcium
observed must be representative of the in vivo distribution of the ion. We have previously shown that the first
requirement can be satisfied by quick freezing followed
by freeze drying (McGuffee et al., 1979; McGuffee et al.,
1981; McGuffee et al., 1985).We have also examined the
extraction of calcium during tissue processing (McGuffee et al., 1981; McGuffee and Little, 1986). Those
studies indicate that extraction of calcium is greatly
reduced in freeze-dried tissue compared to conventionprepared tissue. The
that is
can
be associated with the removal Of extracellular 01 SIXface calcium. We must emphasize that redistribution
0 1987 ALAN R. LISS, INC.
within the tissue has not been measured directly. However, if tissue processing induces a significant redistribution of calcium, it would not be possible to associate
changes in calcium localization with a specific physiological treatment.
To further define the usefulness of EM 45Ca ARG in
studying calcium in smooth muscle, we have begun to
examine the distribution of calcium under different
physiological conditions. As part of a previous paper
(McGuffee et al., 1985), we compared the distribution of
developed 45Ca grains in the vas deferens of the guinea
pig with that of the rabbit. Prior to freezing, the guinea
pig tissue was rinsed for 6 min in nonradioactive physiological salt solution (PSS), whereas the rabbit tissue
was not rinsed. These two tissues differed significantly
in their relative calcium distribution in the cytoplasmic
matrix, plasma membrane, and mitochondria. However,
we could not determine whether the difference was due
to species differences, to the rinse in nonradioactive PSS
prior to freezing, or to both. In this paper we present the
results of studies designed to sort out these differences.
We also report the results of morphometric analyses of
the vas deferens from the two species.
MATERIALS AND METHODS
Three male guinea pigs (300-400 gm) were sacrificed
by a sharp blow to the head. The vasa deferentia were
removed and immersed in a physiological salt solution
(PSS) maintained a t room temperature. The PSS had
the following composition in millimoles per liter: NaC1,
125; KCL2.7; CaC12, 1.8; glucose, 11; Tris (Trizma Base;
Sigma Chemical Co., St. Louis, MO) buffer, 23.8. The
Received February 25, 1986; accepted November 13,1986,
Address reprint requests to Linda J. McGuffee, Ph.D., Department
of Pharmacology, School of Medicine, University of New Mexico, Albuquerque, NM 87131.
322
L.J. McGUFFEE, S.A. LITTLE, AND B.J. SKIPPER
solution was adjusted to pH 7.5 with 6N HC1 and was
saturated with 100% oxygen. Tissues were dissected into
approximately 1 mm2 pieces and placed in radioactive
PSS that contained 45CaC12 (1 mCYml) (total CaCl2 =
1.8 mM) for a period of 65-85 min. At the end of the
incubation period, tissues were rapidly frozen. Tissue
from 3 rabbits was also prepared according to this protocol (McGuffee et al., 1985). Tissue from 3 different
guinea pigs was prepared according to this protocol,
except a 6-min rinse in nonradioactive PSS was added
immediately prior to freezing (McGuffee et al., 1981).
All tissues were quick frozen against a highly polished
copper bar chilled in liquid nitrogen and dehydrated
under vacuum a t low temperature in a glass freezedrying apparatus. The dried tissue was exposed to osmium tetroxide vapor in vacuo and then embedded in
Spurr resin. Detailed descriptions of the freezing, freezedrying, and embedding procedures are given in previous
publications (McGuffee et al., 1981; Chiovetti et al.,
1987).
A thin section through vas deferens cells that were
quick frozen, freeze dried, and embedded is shown in
Figure 1. Comparable tissue fixed in osmium tetroxide
vapor for 2-3 hr, dehydrated in a graded series of ace-
tones (McGuffee and Bagby, 1976), and embedded in
Spurr resin is shown in Figure 2. Ice crystal damage is
minimal in the freeze-dried preparation, and organelles
and membranes are well preserved (compare Figs. 1,2).
Eight blocks of tissue were selected, on the basis of their
degree of morphological preservation, for electron microscopic examination. From each block, 100 nm sections
were cut, placed on copper grids, and coated with a thin
layer of carbon. The grids were then coated with a monolayer of Ilford L-4 emulsion. Following 8-10 wks exposure, autoradiograms were developed in Kodak D-19
developer, stained with uranyl acetate and Reynolds’
lead citrate, and examined in a Hitachi 11-C electron
microscope. Micrographs were taken from grids on which
the background grain density in tissue-free areas of plastic was 20% or less than the grain density over the tissue
(McGuffee et al., 1985).A typical autoradiogram is shown
in Figure 3.
To determine the grain distribution, a circle of 275 nm
radius is drawn around each grain (McGuffee et al.,
1985). If a n organelle or plasma membrane is included
within the circle, the grain is associated with that compound site. For example, the circled grain in Figure 3 is
assigned to the compound site “plasma membrane +
Fig. 1. Smooth muscle of the rabbit vas deferens, quick frozen, freeze dried, exposed to osmium vapor,
and embedded in Spurr low viscosity resin. In this tangential section, part of a nucleus (n) can be seen in
one cell. Extremely electron dense mitochondria (m) appear throughout the cells. Cells are separated from
the extracellular space (e) by a plasma membrane (p). Surface vesicles (arrowheads) are seen along the
membrane. Bar, 1pm.
45CALOCALIZATION IN VAS DEFERENS
323
Fig. 2. Smooth muscle cells of the rabbit vas deferens prepared using conventional techniques, i.e., fixed
in osmium tetroxide vapor, dehydrated in a graded series of acetones, and embedded in S p u n low viscosity
resin. The cells are separated from one another by extracellular space (e). Clusters of surface vesicles
(arrowheads) are evident at discrete sites along the plasma membrane (p). Within the cell, mitochondria
(m) and clear, membrane-bound spaces, presumably sarcoplasmic reticulum (s), are observed. Bar, 1 pm.
cytoplasmic matrix + extracellular space.” The next
step is to estimate the relative area of each component
of the compound site. Using the above example, the area
of the circle occupied by membrane, matrix, and extracellular space is determined from the morphometric
analysis.
From grain-counting and morphometric data, we calculate the grain density of the matrix farther than 275
nm away from the plasma membrane and organelles.
Likewise, we calculate the grain density of the extracellular space away from the plasma membrane. If’ we
assume that the grain density of the matrix inside the
circle is the same as the grain density in the matrix
away from the circle, we can estimate what proportion
of the grains observed close to the membrane originated
from the membrane and what proportion originated from
the matrix. A similar determination is made for the
extracellular space. The relative activity of the membrane can then be determined.
Statistical Analysis
Because of the relatively high energy (mean energy =
0.07 Mev; maximum energy = 0.25 Mev) of the beta
particle emitted during 45Ca decay, a developed grain
will frequently be observed outside of its source. This is
especially true when the source of radiation is small
relative to the range of the beta emission a s in the case
of the plasma membrane, sarcoplasmic reticulum (SR),
and mitochondria. Therefore, it is essential that any
analysis used to evaluate autoradiograms of smooth
muscle take radiation spread into consideration.
Two types of information must be obtained from the
autoradiograms. First, the distribution of 45Ca grains
must be determined and, second, a morphometric analysis of the tissue must be carried out (Salpeter and
McHenry, 1973; Williams, 1969; Williams, 1977). From
these data the relative activity of each site (i.e., proportion of grains associated with the sitelproportion of area
occupied by the site) can be determined. The rationale
and development of the analysis has been described in
detail elsewhere (McGuffee et al., 1981; McGuffee et al.,
1985; Skipper and McGuffee, 1985).
The following example illustrates what relative activity means in terms of our results. Assume that a cellular
site occupies 50% of the area of the tissue. If 50% of the
grains originate from the site, the relative activity is 1.
If 100% of the grains originate from the site, the relative
activity is 2. On the other hand, if a site occupies 1%of
the total area of the tissue, and 5% of the grains are
associated with the site, the relative activity is 5. If 50%
of the grains are associated with the site, the relative
activity is 50. Therefore, we can use relative activity to
answer the question “How is calcium distributed among
the cellular sites?” We cannot, however, use relative
activity to answer the question “What is the millimolar
concentration of calcium in a cellular site?”
324
L.J. McGUFFEE, S.A. LITTLE, AND B.J. SKIPPER
Fig. 3. Smooth muscle of the guinea pig vas deferens incubated in
45Ca, rapidly frozen, freeze dried, osmicated, and embedded in Spurr
low viscosity resin. A portion of a nucleus (n) is seen in one cell.
Extremely electron dense mitochondria (m) are located both peripher-
ally and centrally in the cell. Electron-translucent SR (s) has a similar
distribution. Cells are separated by extracellular space (e).The relative
activity of the organelles, plasma membrane, cytoplasmic matrix, and
extracellular space is determined as described in the text. Bar, 1 pm,
bution of calcium during EM preparation will mask any
physiologically induced changes in calcium distribution.
If, on the other hand, we can associate the localization
of calcium with a physiological stimulus, this would
provide support for the usefulness of the EM ARG technique for studying calcium distribution in smooth muscle. In the nonrinsed guinea pig vas deferens (Table 1,
first column of data), the cytoplasmic matrix and the
mitochondrial distributions are random (i.e., not different from 1.00 [P > .05]). In contrast, there are significantly more grains associated with the SR and the
plasma membrane than would be expected if the distribution were random (P <.001). The nucleus contains
less calcium than would be expected from a random
distribution (P < .05). Thus, there is a selective association of calcium with some (but not all) cellular sites; the
sites of significant accumulation, the plasma membrane
and SR, agree with biochemical data showing calcium
uptake into subcellular fractions containing these organelles (Carsten and Miller, 1980; Daniel et al., 1983;
Raeymaekers
et al., 1983).
RESULTS
In experiments in which the effect of rinsing in nonThe first question we had to address before we could radioactive calcium prior to freezing was investigated,
interpret our results with confidence was, “Does the EM we observed that most of the radioactive calcium is
ARG technique produce significant translocation of cel- removed by a 6-min rinse. The average grain density of
lular calcium?” If translocation is a problem, redistri- the tissue prior to rinsing was 8.6 grainsl.6 pm2. The
Once the relative activity of the cellular sites is determined, the standard error can be estimated using the
methods developed by Skipper and McGuffee (1985).
From examining standard error estimates obtained according to these equations, it becomes obvious that the
smaller the cellular site, the larger the standard error.
The reason for this is that the standard error is inversely
proportional to the proportion of the area of the circle
occupied by that site (see equation 10 in Skipper and
McGuffee, 1985). Thus, the standard error will be large
for a site that occupies 1%of the area, and will be small
for a site that occupies 90% of the area.
For the morphometric analysis, a standard point
counting method was used to estimate the relative (i.e.,
fractional) volume occupied by each of the cellular sites
(Weibel and Bolender, 1973). A double lattice test grid
with scale grid spacing of 275 nm was superimposed on
a micrograph (McGuffee et al., 1985). The number of
points falling on each cellular site was counted and
summed from all the micrographs.
45CA LOCALIZATION IN VAS DEFERENS
TABLE 1. Relative activities of cellular sites in nonrinsed
325
From the data we were also able to estimate the relaand rinsed guinea pig vas deferens
tive volume of the cell occupied by organelles, the plasma
Nonrinsed relative Rinsed relative'
Z
membrane, and the cytoplasmic matrix in the two speCellular site
activitv f SE
activitv + SE value2 cies (Table 3). Chi square analysis indicates that the two
species differ in the volume fraction occupied by the
Total grains
2,853
277
cellular
constituents. In particular, the nuclear volume
Cytoplasmic
0.98 f 0.02**
0.77 f 0.06
3.43
fraction is larger in the rabbit than in the guinea pig,
matrix
and accordingly the cytoplasmic matrix is greater in the
Plasma
2.60 f 0.45**
11.30 f 1.90
-4.45
guinea pig than in the rabbit.
membrane
Mitochondria
0.31 f 0.66*
8.26 f 2.73
-2.83
DISCUSSION
SR
3.87 f 0.46
4.38 f 1.87
-0.26
Nucleus
0.73 f 0.11
1.18 f 0.40
-1.06
We have previously published the results of 45Ca EM
ARG studies that were carried out on freeze-dried vas
'Data from McGuffee et al., 1985.
2Significance levels for z value:
deferens of the guinea pig and the rabbit (McGuffee et
I Z 1 Q 1.96 then N.S.
al., 1985). Major differences in calcium localization were
2.58 < 1 Z I ~ 3 . 2 ,001
9
< P < .01
observed,
but, because the experimental conditions prior
3.29 < I Z I P < ,001.
to quick freezing of the tissues were not identical in the
*P < .01.
**P < ,001.
two studies, we were unable to fully evaluate the differences. Thus, we could only speculate as to whether the
observed distributions were due to rinsing in nonraaverage grain density after rinsing was 2.11.6 pm2. This dioactive calcium, due to species differences, or due to
represents a decrease in grain density after rinsing of both. The present study was designed to sort out these
possibilities.
about 75%.
In interpreting studies that examine the effect of rinsThe next step was to determine whether the grain
distribution was the same after rinsing as it was before. ing on calcium distribution, one must remember that
One would expect that if grains are removed uniformly the concentration of calcium per se in the external mefrom sites of localization, the relative activity of each dium is the same in both the incubation and the rinse
site will remain the same after rinsing, even though the solution. The difference between solutions is that radiototal grain density has decreased. On the other hand, if active calcium in the incubation solution has been rea site decreases in relative activity after rinsing, this
would indicate that the rate of removal is nonuniform
and that calcium in this site exchanges more rapidly TABLE 2. Relative activities of cellular sites in nonrinsed
than does calcium in sites where there is no change in
guinea Dip. and nonrinsed rabbit vas deferens
relative activity. The converse is true if the relative
Z
Guinea pig relative Rabbit relative'
activity of a site increases.
activity f SE
activity f SE value2
Grain distributions with and without rinsing in non- Cellular site
radioactive calcium for 6 min are given in Table 1. The Total grains
2,853
1,180
0.98 f 0.02*
1.05 & 0.03
2.24
relative activities of the plasma membrane and the mi- Cytoplasmic
tochondria were significantly higher after rinsing; the matrix
2.27 f 0.49
-0.05
2.60 f 0.45
cytoplasmic matrix was significantly less radioactive Plasma
after rinsing; the SR and the nucleus were not signifi- membrane
0.31 f 0.66
2.05 k 0.90
1.55
cantly different after rinsing. This suggests that radio- Mitochondria
2.02 f 0.92
-1.81
3.87 f 0.46
SR
active calcium in the cytoplasmic matrix exchanges most Nucleus
0.73 0.11
0.69 f 0.10
-0.31
rapidly with nonradioactive calcium, SR and nuclear
calcium exchange less rapidly, and plasma membrane 'Data from McGuffee et al.. 1985.
'Significance levels for z value:
and mitochondrial calcium exchange least rapidly.
I Z I g 1.96 then N.S.
Next, we compared the distribution of calcium in non- 1.96 < I Z I g 2.58 .01 < P < .05.
rinsed vas deferens from the rabbit (McGuffee et al., *P < .05
1985) and the guinea pig. The results of the analysis are
shown in Table 2. When plasma membrane from the TABLE 3. Volume of cellular sites as a percentage of total
guinea pig was compared with plasma membrane from
cellular volume in the vas deferens
the rabbit, the relative activity was not different. SimiCellular
site*
Guinea pig %
Rabbit%
larly, the relative activity for mitochondria, SR, and the
nucleus from guinea pig were not different from the Cytoplasmic
90 (6298)'
82 (3173)
relative activity of the same organelle in the rabbit. This matrix
suggests that under identical physiological conditions, a Plasma
3 (204)
4 (171)
membrane
given organelle or the plasma membrane has similar
1(83)
2 (68)
calcium sequestration properties in both species. In con- Mitochondria
3 (212)
2 (73)
trast, the distribution of calcium in the cytoplasmic ma- SR
Nucleus
3
(241)
10 (381)
trix was different in the two species. The matrix of the
100 (7038)
100 (3866)
guinea pig had significantly less calcium associated with Tntal
it than did the matrix of the rabbit. However, given the *Overall chi square = 231.67 (P < .001).
absolute magnitude of this difference, the physiological 'Figures in parentheses indicate number of points falling over each
cellular site.
significance is questionable.
326
L.J. McGUFFEE, S.A. LITTLE, AND B.J. SKIPPER
placed with nonradioactive calcium in the rinse solution.
Thus, the overall number of 45Ca grains that we visualize with ARG should decrease after rinsing as nonradioactive calcium replaces radioactive calcium in the
cell. This is, in fact, what we observed.
We also observed a significantly different pattern of
distribution of the 45Cathat remained in the tissue after
rinsing. For example, rinsing caused a 21% decrease in
the relative activity of the cytoplasmic matrix (Table 1).
This suggests that there is a pool of calcium in the
matrix that is accessible to extracellular calcium and
that turns over more rapidly than does the calcium
associated with other sites. Presumably the calcium that
remains in the matrix after rinsing is bound to proteins.
The identity of these proteins is unknown; however, by
use of X-ray microanalysis, Bond et al. (1984b) have
postulated the existence of high-affinity calcium-binding
proteins in the matrix that may act a s calcium buffers
in vascular smooth muscle. Our data suggest that similar binding proteins may exist in the vas deferens. The
fate of the calcium that is removed by rinsing is undetermined. However, it is not unreasonable to assume
that it moves from the matrix to the plasma membrane.
From the plasma membrane it can be removed from the
cell by calcium extrusion mechanisms (Daniel et al.,
1983; Van Breemen et al., 1980).
There is a selective association of calcium with the
plasma membrane in all of our experiments. This is in
agreement with studies that have shown calcium uptake into enriched membrane fractions (Daniel et al.,
1983; Kwan et al., 1983; Raeymaekers et al., 1983). In
addition, this selective association increases after rinsing. An increase in relative activity of the plasma membrane after rinsing may indicate that this pool of calcium
exchanges slowly with the extracellular calcium. Alternatively, it may be a pool that exchanges readily with
extracellular calcium, but is continuously replenished
with radioactive calcium released from internal sites
such as the cytoplasmic matrix. In either case, these
data support the concept that the plasma membrane
plays a major role in cell calcium regulation.
Mitochondria, isolated from a variety of different
smooth muscles, can accumulate calcium in a n energy
dependent manner (Batra, 1982; Ford and Hess, 1975;
Janis et al., 1977). However, mitochondrial uptake of
calcium does not appear to play a major role in regulating cytoplasmic calcium a t concentrations below about
1pM (Daniel et al., 1983; Ford and Hess, 1975; Janis et
al., 1977; Johansson and Somlyo, 1980). In our studies,
the free internal calcium concentration should be well
below 1 pm since the cells have not been exposed to a n
agonist to induce contraction (Filo et al., 1965). Consequently, mitochondria are not likely to act as a n important calcium sink under these conditions. On the other
hand, the increase in the relative activity of the mitochondria after rinsing may indicate the presence of a
slowly exchanging pool of calcium. This explanation is
consistent with our data and with the hypothesis that
mitochondrial calcium is relatively inaccessible a t physiological calcium concentrations.
As with the plasma membrane, there is a selective
association of calcium with the SR in both the guinea
pig and the rabbit. Unlike the plasma membrane, there
was no significant difference in the relative activity of
the SR before or after rinsing. This suggests that the SR
does not sequester calcium as tightly as do the mitochondria, nor does it turnover a s rapidly as does matrix
calcium. The SR seems to function as a n intermediately
exchangeable calcium pool. This observation is consistent with the idea that SR calcium may play a role in
contraction (Bond et al., 1984a,b;Johansson and Somlyo,
1980).
The nucleus does not accumulate calcium, nor does
rinsing significantly alter the relative activity of this
organelle in the guinea pig. Furthermore, the guinea
pig nuclear relative activity is not significantly different
from that of the rabbit. The failure of nuclei to sequester
calcium has also been shown in vascular smooth muscle
(Somlyo et al., 1979).
Morphometric analysis indicates that the nuclear volume in the rabbit is less than in the guinea pig. It
follows then that the relative volume of some other
component of the cell must decrease to accommodate
this increase. In the guinea pig vas deferens, this is
accomplished via a reduction in the volume occupied by
the matrix and not by changes in the relative volume of
the organelles. Further study is required to determine
whether there is a physiological significance to the increase in nuclear volume fraction.
When tissues from the rabbit and the guinea pig were
quick frozen without rinsing, the distribution of calcium
was quite similar in the two species (Table 2). The relative activities of organelles and plasma membrane were
not significantly different between species. The matrices
in the two species were slightly different, but given the
small magnitude of this difference, physiological relevance is doubtful.
In summary, we have demonstrated that the relative
distribution of calcium in the cell can be altered by physiological means. Thus, our results are not consistent with
significant translocation of calcium during tissue processing. The data indicate that the same cellular sites
that showed significant changes when we compared the
vas deferens of the rabbit (nonrinsed) and the guinea pig
(rinsed) are also significantly different when we compare
the nonrinsed and rinsed guinea pig vas deferens. This
indicates that species does not play a major role in
determining the observed distribution of calcium in vas
deferens from rabbit and guinea pig. The relative distributions before and after rinsing show several significant
differences, indicating that the rate of calcium turnover
is not uniform among the cytoplasmic matrix, plasma
membrane, mitochondria, SR, and nucleus.
ACKNOWLEDGMENTS
This work was supported by National Institutes of
Health grant 1-R01-GM30003. L.J.M.’s work was done
during the tenure of a n Established Investigatorship
from the American Heart Association and with funds
contributed in part by the New Mexico affiliate.
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