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Effects of temperature and buffer composition on calcium sequestration by sarcoplasmic reticulum and plasma membrane of rabbit renal artery.

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THE ANATOMICAL RECORD 228:288-296 (1990)
Effects of Temperature and Buffer Composition
on Calcium Sequestration by Sarcoplasmic
Reticulum and Plasma Membrane of Rabbit
Renal Artery
Departments of Pharmacology (L.J.M., S.A.L., J.V.M.) and Family, Community and
Emergency Medicine (B.J.S.) School of Medicine, University of New Mexico, Albuquerque,
New Mexico 87131; Department of Pharmacology, School of Pharmacy, University of
Wisconsin, Madison Wisconsin 53706 (E.S.W.-C.)
45Ca electron microscopic autoradiography was used to examine
the effects of buffer composition and temperature on the distribution of calcium in
rabbit renal artery smooth muscle cells. The results show that the relative distribution of calcium is dependent on both the buffer used (Tris or Krebs) and the
temperature of the bathing solution (25°C or 34°C). Krebs buffer at 34°C gave the
highest relative activity in the plasma membrane, sarcoplasmic reticulum, and
mitochondria. Buffer and temperature had little effect on the relative activity of
the nucleus or cytoplasm. Next, we identified the cellular sites of calcium accumulation after 5, 15, 30, or 60 min exposure to 45Ca in Krebs buffer at 34°C. The
results show that sarcoplasmic reticulum and plasma membrane are the primary
sites of calcium accumulation during influx into these cells. Although the amount
of 45Ca in the cell continues to increase with longer exposure, the relative distribution of calcium is essentially the same after 5 or 60 min. The data also indicate
that the relative activity of plasma membrane + sarcoplasmic reticulum (a combination site that includes sarcoplasmic reticulum within a mean distance of 275
nm of the plasma membrane) is similar to the membrane alone and is lower than
the sarcoplasmic reticulum alone.
Several laboratories have reported on the suitability
of Tris buffer for studying contractile responses in
smooth muscle. Although some investigators found
that Tris has a significant depressant effect (Altura et
al., 1980; Sakai et al., 1983; Turlapaty et al., 1978),
others reported that contractile responses are not inhibited in Tris solutions (Hayashi and Hester 1987;
Johansson et al., 1979; Karaki et al., 1981).In addition,
temperature (25°C vs. 34°C) has been reported to affect
smooth muscle contractility (Karaki et al., 1987). In
1986, Wheeler-Clark et al. (1986) reported significant
45Ca accumulation in the plasma membrane and sarcoplasmic reticulum of rabbit renal artery in the absence of pharmacological stimulation. This accumulation is two to five times greater than previously
reported in guinea pig and rabbit vas deferens
(McGuffee et al., 1985, 1987). The latter studies were
conducted at room temperature (25°C) and in Tris buffered (23.8 mM) solution. The higher temperature
(34°C) and lower concentration of Tris (6 mM) used by
Wheeler-Clark et al. (1986)were suspected to be major
contributors to the increase in plasma membrane and
sarcoplasmic reticulum calcium observed in that study.
However, tissue and species differences could not be
ruled out. With this in mind, we designed experiments
to define better the effect of buffer and temperature on
the cellular distribution of calcium in rabbit renal ar0 1990 WILEY-LISS, INC.
tery smooth muscle. In the present paper, we present
data obtained at 25°C and 34°C in Krebs Ringer bicarbonate buffer and at 25°C in 23.8 mM Tris. We have
examined these muscles using in vitro tension measurements and 45Ca electron microscope (EM) autoradiography (ARG), which we previously have shown has
sufficient ultrastructural and spatial resolution to localize calcium within smooth muscle cells (McGuffee et
al., 1985, 1987; Skipper and McGuffee 1985; WheelerClark et al., 1986).
Calcium flux studies in intact smooth muscle tissue
have shown that equilibrium between calcium influx
and efflux is reached in about 60 min (Hester e t al.,
1978). However, flux studies cannot identify sites of
calcium accumulation within the cell. Moreover, they
cannot be used to determine whether, over time, calcium redistributes among cellular sites. This information can only be obtained with a technique that combines ultrastructural resolution with calcium flux. In
the present study, we used EM ARG to identify the
cellular sites that sequester calcium after 5, 15, 30, or
60 min of exposure to 45Ca. Some of these results were
reported previously (McGuffee et al., 1988).
Received January 8, 1990; accepted March 2, 1990.
Krebs Ringer bicarbonate solution (KRB) contained
125.1 mM NaC1, 4.7 mM KCl, 1.2 mM KH,PO,, 18.7
mM NaHCO,, 1.2 mM MgS0,.7H2O, 2 mM CaCl,, 5.6
mM glucose. A depolarizing KRB solution was made by
replacing Na+ with K + , giving a final K + concentration of 129.1 mM. All KRB solutions were oxygenated
with 95% 0, and 5% CO,. Solutions were adjusted to
pH 7.4 a t the temperature at which they were to be
used (25°C or 34°C). The Tris solution contained: 125
mM NaC1, 2.7 mM KC1, 2 mM CaCl,, 11 mM glucose,
and 23.8 mM Tris (Trizma Base, Sigma Chemical Co.,
St. Louis, MO) buffer. A depolarizing Tris solution was
made by replacing Na+ with K + in the high Tris solution, giving a final K + concentration of 127.7 mM. All
Tris solutions were oxygenated with 100% O,, and the
pH was adjusted to 7.5 at either 34°C or 25°C.
Tension Studies
Renal arteries were removed from six male NZW
rabbits, immersed in oxygenated KRB solution,
cleaned of connective tissue, and cut into spiral strips.
Each spiral strip was cut in half, giving a total of four
tissues per animal. Strips were hung in muscle baths
under 500 mg of tension, and equilibrated in oxygenated solution for 90 min. The equilibration solution
was one of the following: KRB at 34”C, KRB at 25”C,
23.8 mM Tris at 34“C, or 23.8 mM Tris at 25°C. The
solution was then drained from the bath and replaced
with the similar K + substituted depolarizing solution
for 10 min to induce contraction. The depolarizing solution was drained and replaced with one of the three
remaining nondepolarizing solutions, and the sequence
was repeated. I n this manner, muscles were exposed to
all four experimental conditions in a randomized order.
The magnitude of each contraction was measured
and a two-way ANOVA using specimen and solution as
the independent variables was used to test for significant differences among treatments. All possible pairwise comparisons among treatments were made using
the Newman-Keuls procedure (SAS, 1988).
45Ca EM Autoradiography Studies
Temperature and buffer solution studies
Renal arteries were removed from three male NZW
rabbits and placed in oxygenated KRB solution. Arteries were cleaned of connective tissue, cut into 2 mm
ieces, and incubated in oxygenated KRB containing
‘%a (1mCi/ml, total Ca = 2.0 mM) for 60 min either
a t room temperature (25°C) or at 34°C. Other tissues
from the same renal arteries were incubated in Trisbuffered solution (23.8 mM) containing 45Ca at room
temperature. After 60 min, tissues were quick frozen
and freeze-dried as described below.
45Ca uptake vs. time studies
Renal arteries from three male NZW rabbits were
incubated in oxygenated KRB containing 45Ca (1mCi/
ml, total Ca = 2.0 mM) a t 34°C. Tissue from each rabbit was exposed to 45Ca for 5, 15, 30, or 60 min. After
the appropriate exposure period, tissues were quick frozen and freeze-dried as described below.
Quick freezing, freeze-drying, and
preparation of autoradiograms
Tissues were quick frozen against a highly polished
copper bar using the ‘“Gentleman Jim” freezing device
(Ted Pella, Inc.). Frozen tissues were maintained below
- 80°C throughout the drying procedure to prevent formation of liquid water within the specimens (Chiovetti
et al., 1987). Tissues were dehydrated under vacuum a t
low temperature in a glass freeze-drying apparatus
(Chiovetti et al., 1987; McGuffee et al., 1981). The dried
tissues were exposed to osmium tetroxide vapor in
vacuo and embedded in Spurr resin. An example of the
quality of tissue preservation with this technique is
given in Figure 1. Tissues were osmicated in vacuo to
prevent subsequent calcium loss from the freeze-dried,
Spurr-embedded smooth muscle when the tissue is cut
on water (McGuffee and Little, 1986). Thin (90-100
nm) sections were cut onto water using a Sorvall MT2B ultramicrotome, collected on EM grids, and carbon
coated (Chiovetti e t al., 1987).
Based on the ultrastructural preservation of the
cells, eight blocks of tissue were selected from each
treatment, and a minimum of six grids of tissue were
collected from each block. Grids were coated with a
monolayer of Ilford L4 emulsion according to the
method described by Caro and Van Tubergen (1962).
Briefly, this method involved melting the emulsion in
a water bath then cooling the emulsion a t room temperature. To determine when the emulsion is a t the
correct temperature to form a monolayer, a 4-cm-diameter wire loop was dipped into the emulsion and
withdrawn at a uniform rate. The thin film in the loop
was then touched to the surface of a microscope slide to
deposit the film on the slide. When dry, a uniform
monolayer of Ilford L4 emulsion has a purple interference color (Salpeter and Bachmann, 1972). Once a
monolayer was obtained, slides containing the grids
were coated. The emulsion was exposed in the dark a t
4°C for a n appropriate period (8-12 weeks). Autoradiograms were processed in Kodak D-19 developer and
Fixer and in-cubated in Reynolds’ lead citrate for 2030 min to remove undeveloped emulsion. Grids were
poststained in saturated uranyl acetate in 50% ethanol
followed by Reynolds’ lead citrate to enhance contrast.
Exposure to neither uranyl acetate nor Reynolds’ lead
citrate causes a shift in the location of developed silver
grains (McGuffee et al., 1981).
Micrographs were taken only from grids that had a
background grain density in tissue-free areas of plastic
of 520% of the grain density over the tissue. Micrographs were taken at a n initial magnification of 10,000
and photographically enlarged to 24,000 for morphometric and grain distribution analyses. A typical autoradiogram is shown in Figure 2.
Data collection and analysis
Our goal is to associate the developed grains with
their cellular origin. The sites of origin in which we are
interested are the cytoplasmic matrix (C), which occupies most of the cell volume, and the plasma membrane
(PM), nucleus (N), sarcoplasmic reticulum (SR), and
mitochondria (MI, which occupy much smaller propor-
Fig. 1. Rabbit renal arteries were rapidly frozen, dehydrated under vacuum below -9O"C, exposed to
osmium tetroxide vapor to enhance contrast and to preserve ionic distribution, embedded in Spurr resin,
and thin sectioned onto water. The ultrastructure of the cells is similar to conventionally prepared tissue,
with the exception of mitochondria (M), which are extremely electron dense in freeze-dried tissue. The
nucleus (N) and sarcoplasmic reticulum (S) are well preserved. Cells are separated from one another by
extracellular matrix (E). Bar = 1 p,m.
tions of the cell. Due to the relatively high energy
(mean energy 0.07 MeV; maximum energy = 0.25 MeV)
of the p particle emitted during 45Cadecay, 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, as in
the case for PM, SR, and M. We account for radiation
spread with the following analysis.
Morphometric data are obtained from the autoradiograms using the point and circle technique described
by Williams (1969,1977). Grain density information is
obtained using the probability circle method of Salpeter and McHenry (1973). The statistical analysis
that allows us to compare data from different treatments was developed in our laboratory (Skipper and
McGuffee, 1985).
To determine the grain distribution, a 275nm-radius circle is centered over each grain (McGuffee
et al., 1985). Our choice of this size circle is based on
both theoretical and experimental data showing that
275 nm approximates the half distance for a point
source of calcium (Salpeter et al., 1987; Winegrad,
Fig. 2. A tangential section was cut through renal artery cells after incubation with 45Ca,freeze drying,
and preparation for EM ARG. Thirteen 45Ca grains are shown on this autoradiogram. A 275 nm radius
circle is drawn around one of the grains, which overlies the compound site “PM C + EM.” The method
for partitioning grain density among PM, C, and E in this compound site is discussed in Data Collection
and Analysis. Abbreviations as in Figure 1. Bar = 1 Km.
1965). If a n organelle or PM is included within the
circle, the grain is assigned to the respective compound
site. For example, the circled grain in Figure 2 is assigned to the compound site “PM + C + E.” As can be
seen in the figure, PM never fills a 275-nm-radius circle. It is always observed a s part of a compound site.
The next step is to estimate the fractional volume of
PM, E, and C in the compound site. This is done by
placing a plastic overlay containing a n array of 275nm-radius circles, the center of each circle denoted by a
point, on each autoradiogram (Wheeler-Clark et al.,
1986). For circles overlying PM + C + E, the individual site (PM, C, or E) under the point is recorded. By
repeating this process on all the autoradiograms, the
fractional volume occupied by each component of PM +
E is calculated (Weibel and Bolender, 1973).
The final step is to calculate the grain density of PM.
From other data we have already calculated the grain
density of C more than 275 nm away from organelles
and PM. We also have calculated the grain density for
E. We must assume that the grain density of C included in PM + C + E is the same as the grain density
of C observed alone a s a n individual site. A similar
assumption is made for the E. We are unaware of any
evidence in the literature t h a t would negate these assumptions. We then subtract the grain density contributed to PM + C + E by C and by E. The remaining
grain density is allocated to PM.
Autoradiographic data are expressed in units of relative activity:
Relative activity
where Gi is number of grains observed in a cellular
site, Ai is the fractional volume for that site, GT is the
total number of grains counted, and AT is the total
volume. [Note: The method we used to calculate relative activity is based on the equation published by
Williams (1977) and Kent and Williams (1974) to calculate what they called “relative crude specific activity.”]
Estimates of relative activity can be obtained for PM,
SR, M, and N a s long as they are surrounded by C
and/or E. When two small sites, for example, PM and
SR, are included in one compound site, additional equations would have to be used to attribute activity proportionately. Although we have not yet developed
these equations, we predict that the variability estimates calculated with these new equations would be
very large relative to the mean relative activity. Moreover, additional assumptions (i.e., homogeneity of activity among central and peripheral SR) would have to
be made. However, if PM + SR is considered as one cellular site separate from PM or SR, the technique can be
used to estimated the relative activity of the PM + SR
combination site. The same is true for any other combination site.
The following example illustrates what relative activity means in terms of our results. If the SR occupies
4% of the cell and contains 4% of the grains, the relative activity is 1. If 40% of the grains are in the SR, the
relative activity is 10. If the N occupies 8% of the cell
and contains 4% of the grains, the relative activity is
0.5. If 40% of the grains are in the N, the relative
activity is 5. Thus relative activity estimates the distribution of cellular calcium among cellular components. It does not estimate the molar concentration of
calcium. A relative activity of >1.0 indicates calcium
accumulation by the site; a relative activity of <1.0
indicates calcium exclusion by the site. A relative activity = 1.0 indicates no accumulation or exclusion by
the site. [Note: In previous publications (McGuffee et
al., 1985, 1987; Wheeler-Clark et al., 19861, we have
called this a random distribution.]
As discussed in Skipper and McGuffee (19851, the
calculation for relative activity assumes no variability
in the area measurement (Ail for the cellular sites.
Although this assumption does not appreciably affect
the results for sites that occupy a relatively large
portion of the cell such as C, PM, and N it is not valid
for sites that occupy less than about 5% of the cell and
have a high relative activity (Skipper and McGuffee,
1986). The net result is that the standard error of the
mean (SEM) calculated from the equations in Skipper
and McGuffee (1985) for a large site is a more reliable
estimate of variability than is the SEM calculated for a
small site. To obtain a better estimate for the SEM of
all cellular sites, we have developed a simulation program that introduces variability in Ai (Skipper and
McGuffee, 1986). We have used this computer program
to determine the SEM for each experimentally determined value of relative activity reported in this paper.
All possible pairwise comparisons of relative activities for each cellular site were made using a Z test
(Skipper and McGuffee, 1985). Because four different
treatments were compared for each site, the upper
limit for P was set at 0.008 (a = 0.05/6 Bonferroni
correction) for determining significance (Duncan e t al.,
TABLE 1. Contractile remonses
generated in KRB or 23.8 mM-Tris buffer
at 25°C or 34°C’
KRB 34°C
KRB 25°C
Tris 34°C
Tris 25°C
Grams of tension
(mean t 2 SEMI
2.87 i 0.43*
2.02 t 0.39
2.54 i 0.39*
1.95 t 0.35
*Significantly different from all other treatments
( P < .01).
*n = 22 tissues for each treatment; KRB = Krebs
Ringer bicarbonate buffer.
In vitro tension studies were carried out to examine
the effects of buffer type and temperature on the rabbit
renal artery’s capacity to contract. The results are
shown in Table 1. Temperature of the bathing solution
had a greater effect on the amount of tension produced
by the tissue than did buffer composition. Tissues generated the greatest contractile tension in KRB at 34”C,
followed by Tris at 34”C, KRB at 25”C, and Tris at 25°C.
Using the Newman-Keuls procedure, all possible pairwise comparisons were examined for significant differences. Both KRB at 34°C and Tris at 34°C were significantly different from each other and from KRB and
Tris at 25°C (P < .01). KRB and Tris a t 25°C were not
significantly different from each other.
The fractional cell volume occupied by the cellular
sites was determined in tissues from three animals
that had been incubated in KRB at 34°C. On average,
76.5% of the cell is C, 8.7% PM, 7.9% N, 4.4% SR, 2.0%
M, and 0.5%PM + SR. As is discussed below, sites that
occupy less than about 5% of the cell will have the
largest SEM relative to the magnitude of the relative
We used EM ARG to examine the effects of buffer
solution and temperature on calcium distribution
among the PM, SR, M, N, and C. Results are expressed
in units of relative activity and are given in Figure 3.
The relative distribution of calcium is dependent on
both the buffer (Tris or KRB) and the temperature
(25°C or 34°C). Use of KRB at 34°C gave the highest
relative activity in the PM, SR, and M. The data in
Figure 3 shows that there is calcium sequestration at
the PM in KRB at 25°C and 34°C (relative activity
significantly >1.0) but not in Tris at 25°C. Pairwise
comparisons of the three different treatments were carried out using a Z test. The relative activities of PM in
KRB at 25°C and 34°C (columns 2 and 3) are not significantly different from one other. This suggests that
45Cauptake at the PM is more sensitive to the type and
concentration of buffer than to temperature. The relative activity in 23.8 mM Tris buffer at 25°C (column 1)
is significantly less than with either of the other treatments.
In contrast to the PM, sequestration of calcium a t the
SR depends more on the temperature of the bathing
medium than on the type of buffer. There is calcium
sequestration in the SR in KRB a t 34°C (relative activity significantly >1.0) but not a t 25°C. In KRB a t
34°C (column 3 in Fig. 3),the relative activity of the SR
is significantly greater than at 25°C in either Tris or
.3 12
0TRIS, 25°C
KRB, 25°C
KRB, 34°C
0 5 rnin
= 15
30 rnin
60 rnin
a , E
z 4
Cellular Sites
Fig. 3. Relative activity is shown for each cellular site in the rabbit
renal artery in the presence of either Tris or KRB a t 25°C or 34°C.
Data for Tris buffer at 34°C are from Wheeler-Clark et al. (1986).
(Note: in Figs. 3 and 4 and Table 1, mean values & 2 SEM are plotted,
since 2 SEM represent the 95% confidence interval.) All possible pairwise comparisons were made for each tissue site. Relative activities
that are significantly different ( P < .05) are PM: 1,2; 1,3; SR 1,3; 2,3;
N: 1,3;C: 1,2; 1,3.
KRB (columns 1 and 2). This suggests that a greater
fraction of the total cellular calcium is in the SR at
34°C than at 25°C.
There is no significant calcium sequestration in the
M with any treatment (relative activity not significantly different from 1.0). The relative activity of the M
is higher at 34°C (column 3) than at 25°C (column 1and
2). However, none of the three treatments are significantly different from one another due to large variability in relative activity estimates. As was discussed in a
prior publication (Skipper and McGuffee, 19851, the
SEM is inversely proportional to the fractional volume
of the site. Morphometric results indicate that M occupy about 2% of the rabbit renal artery cell. Consequently, the SEM of the relative activity for M will be
large. Thus it will be very difficult to detect statistically significant shifts in calcium associated with M
unless unrealistically large sample sizes are analyzed.
Type of buffer and temperature have very little effect
on the relative activity of N and C. There is no significant sequestration of calcium in the N or C in any of
the treatments (relative activity I1.0). However, N
relative activity is significantly greater in Tris buffer
at 25°C than in KRB at 34°C (columns 1 and 3 in Fig.
3). There is significantly more calcium in the C in Tris
at 25°C than in KRB at 25°C (columns 1 and 2). These
results suggest that more of the total cellular calcium
is in the N and C in Tris buffer at 25°C than in KRB at
34°C. In addition, the C has more of the total cellular
calcium in 23.8 mM Tris buffer at 25°C than in KRB at
Figure 4 shows how 45Ca is distributed among the
cellular sites after 5, 15, 30, and 60 min exposure to
45Ca in KRB at 34°C. These data indicate only the fractional distribution of the total 45Cauptake at each time
interval and the reader should be reminded that the
total 45Ca accumulation has been shown to increase
over time up to 60 min (Hester et al., 1978). Radioac-
Cellular Sites
Fig. 4. Relative activity 2 2 SEM for cellular sites after varying the
time of exposure to 45Cain the bathing medium. All possible pairwise
comparisons were made for each tissue site. Relative activities that
are significantly different ( P < .05) are PM: 2,3; 3,4; SR: 3,4. The
relative activity for M at 5 and 30 min is below, but not significantly
different from, zero. Because of random variability and the small volume fraction occupied by M in this tissue, it is mathematically possible to calculate a relative activity value below zero. However, negative values are physiologically meaningless and should be
considered zero.
tive calcium is detected in all the cellular sites at every
time period. However, the distribution of labeled calcium is not uniform across cellular sites, nor does the
distribution remain constant with time.
Within 5 min, calcium sequestration is observed at
the PM. Sequestration is also observed after 15 and 60
min (relative activity significantly >1.0) but not after
30 min. The relative activity after 30 min (column 3) is
significantly less than that after 15 or 60 min (columns
2 and 4) but not significantly different from that at 5
min (column 1).
The SR sequesters a major proportion of the total
cellular calcium within 5 min. The SR continues to
sequester calcium after 15, 30, and 60 min (relative
activity significantly >1.0). The only significant shift
in SR calcium between 5 and 60 min is a decrease after
30 rnin compared to 60 min.
In addition to examining PM and SR alone, we estimated the relative activity of the combination site PM
+ SR. PM + SR includes junctional and peripheral SR
within 550 nm of the PM (mean distance = 275 nm). In
this study, PM + SR does not sequester significant
levels of calcium at any time period (relative activity
not significantly different from 1.0) and relative activities after 5, 15, 30, and 60 min are not significantly
different from each other. Calcium is not sequestered
by the C a t any time period (relative activity <1.0), nor
are there significant differences in relative activity
among the four time periods. The same is true of the N
and the M.
Contractile responses in vascular smooth muscle are
temperature dependent. Significantly more tension is
produced by strips of rabbit renal artery in KRB or in
Tris at 34°C than at room temperature (25°C) (Table 1).
A similar observation was made by Karaki et al.
(1987), who compared contractility of rabbit aorta at
37°C and 25°C. Using a bicarbonate-buffered system,
they showed that a t 37°C both norepinephrine and high
potassium elicited a larger increase in tension than at
25°C. Both norepinephrine (Bond et al., 1984; Kowarski e t al., 1985) and high potassium (Wheeler-Clark et
al., 1986) can release calcium from SR. It may be that
we observed a larger contraction at 34°C than a t 25°C
because there is more releasable calcium present in the
SR at the higher temperature. Our EM ARG results,
discussed below, support this hypotheses, since proportionally more calcium is associated with the SR a t 34°C
than at 25°C. A similar observation has been made
using skinned rat caudal artery. In that preparation,
there is over 75% more calcium in the SR at 38°C than
a t 24°C (M. Stout, personal communication).
We examined the effect of buffer composition and
temperature on the distribution of calcium in rabbit
renal artery cells. Results show that the highest relative activities in the PM and SR are obtained when
tissues are exposed to KRB buffer a t 34°C (Fig. 3). The
data also show that 45Ca uptake a t the SR is more
sensitive to the temperature of the bathing medium
than to the type of buffer. Proportionally more calcium
is associated with the SR at 34°C than a t 25°C. This
finding is in good agreement with the results of
Wheeler-Clark et al. (1986). They calculated that at
34°C in 6 mM Tris buffer, a n SR relative activity of
10.5 t 2.0 in this tissue. This is not significantly different from our SR relative activity of 11.6 2 0.9 in
KRB at 34°C (Fig. 3). On the other hand, it is significantly more than the relative activity a t 25°C in 23.8
mM Tris (1.8 -t 0.3) or KRB (2.1 2 0.4) shown in Figure
3. In other words, both 6 mM Tris and KRB at 34°C
support sequestration of calcium in the SR. Neither
23.8 mM Tris at 25°C nor KRB at 25°C support sequestration in the SR.
On the other hand, we found t h a t calcium sequestration at the PM is more sensitive to the type of buffer
than to temperature of the bathing solution. This finding is also in agreement with the results of WheelerClark e t al. (19861, who reported a PM relative activity
of 4.1 t 0.8 in 6 mM Tris at 34°C. This is not significantly different from the relative activity of 4.7 2 0.4
in KRB at 34°C or 4.4 2 0.4 in KRB at 23°C (Fig. 3).
However, it is significantly more than the relative activity of 0.1 2 0.5 in Tris at 23°C (Fig. 3). Thus, in 23.8
mM Tris buffer the PM excludes calcium but in 6 mM
Tris and in KRB i t sequesters calcium.
We conclude from the data in Figure 3 and from the
previous results of Wheeler-Clark et al. (1986) that
both type of buffer and temperature can significantly
alter the relative distribution of calcium in the renal
artery smooth muscle cell. Both are important parameters to consider when designing in vitro experiments
on smooth muscle.
The effect of Tris buffer on isolated smooth muscle
function has been studied in several different laboratories with conflicting results. Turlapaty and coworkers (1978) compared the effects of Tris and KRB buffers
on contractile responses of rat portal vein and aorta.
Tris buffer in a range of 5-30 mM decreased spontaneous mechanical activity in a concentration dependent manner. Tris (5 mM) also attenuated contractile
responses to KC1, epinephrine, and angiotensin. In a
later paper, this same group (Turlapaty et al., 1979)
used calcium flux to study the effect of Tris and KRB
on tissue calcium content and found that total calcium
uptake was reduced in the presence of 30 mM Tris. In
other laboratories, Tris in a range of 5-25 mM had
little or no effect on elicited contractions in rabbit aorta
particularly when the sodium concentration was kept
constant (Karaki et al., 1981). Conflicting results such
a s these have raised questions about the advisability of
using Tris-buffered solutions, particularly in high concentrations, in in vitro studies of contractile tension.
Kwan and Daniel (1981) investigated the effect of
Tris on calcium accumulation by membrane fractions
isolated from rat mesenteric arteries and veins. They
observed that 50 and 100 mM Tris reduced both ATPdependent and -independent calcium accumulation in a
purified PM fraction and that the inhibition was concentration dependent. This suggests that Tris may interfere with both ATP-dependent calcium efflux from
the cells and also with non-ATP-dependent binding to
PM vesicles.
Although previous results suggested that exposure
to Tris might physiologically or biochemically alter calcium accumulation in smooth muscle, they did not
identify unequivocally which calcium pools are affected
by Tris buffer in situ. However, these studies do provide important information on the concentration dependence of a Tris effect and did suggest that a t least
part of the effect is at the PM. The EM ARG data reported here confirm the PM as the primary locus of the
Tris effect in intact tissue. In addition, the effect appears to be concentration dependent, since the relative
activity of the membrane in 23.8 mM Tris is two orders
of magnitude less than the activity in 6 mM Tris
(Fig. 3).
The exact site at which Tris alters calcium binding at
the PM is not clear. As a cationized molecule, Tris
might be acting on the external surface of the membrane to compete with calcium for binding sites. Alternatively, Tris also could interfere with internally
bound calcium, because a significant portion of Tris
(30%) is unionized at pH 7.4 and can penetrate the cell
membrane (Holmdahl and Nahas, 1962). Therefore,
the reduction of PM calcium sequestration by Tris in
intact cells does not necessarily clarify on which side of
the membrane bilayer the PM calcium pool is located.
The mechanism by which Tris inhibits PM calcium
binding is also unclear. Cationic Tris might compete
with calcium for binding to negatively charged proteins, lipids, andlor carbohydrates, such as sialic acid,
associated with the PM-surface coat. In addition, Tris
has been shown to have a variety of effects on animal
and plant proteins (Ljungbery et al., 1984; Murphy and
Sastre, 1983). For example, Tris exposure can alter
membrane phospholipid content of certain bacteria
(Donohue et al., 1982), release outer membrane components (Irvin et al., 1981a), and increase membrane
permeability (Irvin et al., 1981b). Because of the variety and complexity of interactions between membranes
and Tris, it seems equally plausible that the effects
described in this study could result from direct competitive antagonism with calcium for PM binding sites or
from indirect effects on membrane composition and
permeability. It also seems possible that subtle differences in membrane composition could result in spe-
cies-andlor tissue-dependent differences in susceptibility to the effects of Tris. Such differences could
account, in part, for the conflicting results reported for
Tris in the literature. Therefore, additional studies
would be helpful to clarify the mechanism of T r i s
membrane interactions.
Regardless of the manner in which Tris affects membranes, we agree with previous investigators who have
warned against using Tris in high concentrations in
studies of calcium accumulation by isolated membrane
fractions (Kwan and Daniel, 1981; Kwan et al., 1984)
and in intact muscle preparations (Altura et al., 1980;
Sakai et al., 1983; Turlapaty et al., 1978, 1979). In
addition, we suggest that, to obtain maximum uptake
by SR in vascular smooth muscle, experiments should
be carried out at 34°C rather than a t room temperature.
In a separate series of experiments, we examined
calcium sequestration in the PM, SR, PM + SR M, N,
and C after 5, 15, 30, and 60 min exposure to 45Ca in
KRB at 34°C. These results are the first to show that
SR and PM are the primary sites of calcium accumulation during influx into rabbit renal artery. The PM
and SR accumulated significant proportions of the total
cellular calcium after only 5 min; the M, N, and C did
not. Although the amount of 45Ca in the cell continues
to increase with longer exposure (not shown on graph),
the relative distribution is essentially the same after 5
or 60 min. At the present time, we do not have a n
explanation for the significant decrease in the relative
activity at both the PM and SR after 30 min (Fig. 4).
It has been suggested that “peripheral” SR in smooth
muscle can sequester and release large amounts of calcium for excitation-contraction coupling as do the terminal cisernae and junctional SR of skeletal and cardiac muscle. To investigate this possibility, other
investigators have used X-ray microprobe analysis to
identify “hot spots” within 50 nm of the PM in smooth
muscle (Bond et al., 1984). In guinea pig portal vein,
these “hot spots” are presumed to be SR, although SR
has not yet been visually identified as such in these
preparations. Using ARG, we have shown that the relative calcium activity of the PM + SR region is very
close to that of the PM alone and much lower than that
of the “central” SR (Fig. 4). A direct comparison of
these results with those previously published by Bond
et al. (1984) is not possible due to the differences in the
technique. However, our findings show that on average, PM + SR sites in rabbit renal artery do not sequester greater amounts of calcium than the “central”
SR or PM alone. Therefore, the importance of calcium
in the PM + SR region relative to “nonjunctional” PM
or SR in excitation-contraction coupling of smooth
muscle requires further investigation.
This work was supported by NIH grant GM30003 to
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artery, renar, membranes, buffer, sequestration, effect, rabbits, temperature, reticulum, sarcoplasmic, calcium, plasma, composition
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