Aortic actomyosin and spontaneously C. L. SEIDEL
(With
content of maturing hypertensive rats the Technical
Assistance
normal
of R. Bowers)
Department of Medicine, Section of Cardiovascular Sciences, Baylor College of Medicine, Houston, Texas 77030 SEIDEL, C. L. Aortic actomyosin content of maturing normal and spontaneously hypertensive rats. Am. J. Physiol. 237(l):
H34-H39, 1979 or Am. J. Physiol.: Heart Circ. Physiol. 6(l): H34-H39, 1979.-Observations reported in the literature suggest that the maximum contractile response of aorta from spontaneously hypertensive (SHR) rats is reduced compared to that of normotensive controls and that the maximum contractile response of aorta from Wistar-Kyoto (WKY) rats is less than that of aorta from Wistar rats. A possible explanation for these differences is that there are differences in the amount of actomyosin per smooth muscle cell. To test this hypothesis, actomyosin, total protein, and DNA contents of thoracic aorta from 3-, 6-, 12-, and 23-wk-old SHR and WKY rats were determined and compared to similar values previously published by this laboratory for Wistar rats. The absolute amount of actomyosin increased with increasing age in aorta from both WKY and SHR rats, but increased more rapidly in the SHR so that by 23 wk it was significantly greater than that in the WKY. This increase was not correlated with changes in systolic blood pressure during maturation. Sufficient hyperplasia (based on DNA) was observed in the aorta of SHR relative to WKY rats, so that the amount of actomyosin per smooth muscle cell was not different between these two groups at any age studied. Comparison of aortas from SHR, WKY, and Wistar rats indicated that Wistar aortas had more actomyosin and a greater number of cells (based on DNA) per gram of media than aortas from either SHR or WKY rats, but at any age, all three groups had the same amount of actomyosin per smooth muscle cell. These observations suggest that a) the lower maximum contractile response of aorta from SHR rats is not due to a reduction in the amount of actomyosin per smooth muscle cell, b) there are biochemical differences in aorta between subspecies of rats, and c) the WKY rat may be a better control for studies involving SHR rats because the biochemical characteristics of their aorta are similar.
the smooth muscle cell is responsible for the synthesis of collagen and elastin (Zl), it is possible that the synthetic activity of the smooth muscle cell shifts toward the synthesis of connective tissue and away from the synthesis of actomyosin. A reduction in the actomyosin content of the muscle cell could result in a reduction in maximum force-generating ability. One of the objectives of this study was to test this hypothesis by comparing the actomyosin content of thoracic aorta from normotensive and spontaneously hypertensive rats. Second, to determine if changes in cellular actomyosin content were associated with changes in blood pressure, aorta from hypertensive and prehypertensive rats were studied. Third, because it has been demonstrated that in vitro preparations of aorta from Wistar rats develop more maximum tension than similar preparations from WistarKyoto rats (6) raising the question of which animal is the best control for the SHR rat, the present results were compared to previously obtained values for aortas from Wistar rats (23). METHODS
Actomyosin extraction. Male WKY and SHR rats were obtained from Taconic Farms and were allowed to accommodate to their new surroundings for 3-5 days before their systolic blood pressures were determined. Four age groups were studied: 3, 6, 12, and 23 wk. At the time of the experiment, the 3-wk-old rats were anesthetized with ether, and a needle-tipped cannula (23-gauge) was inserted retrograde into the aorta. Pressure was measured with a Statham P23Db pressure transducer and displayed on a Grass model 7 polygraph. The ether was removed and the systolic pressure was noted at the first genetic hypertension; maturation; thoracic aorta; vascular sign of a tactile response. The animal was then immedismooth muscle ately killed by thoracotomy. The systolic blood pressure in all other age groups was determined by the indirect PREVIOUS OBSERVATIONS (23) SUggWt a positive COrrelatail-cuff method and the animals were killed by cervical tion between changes in maximum force-generating abil- dislocation. The thoracic aorta between the subclavian ity of rat aorta and the cellular content of actomyosin. In and celiac arteries was removed from each animal, various animal models of hypertension, many investiga- cleaned of adhering fat, and slit open lengthwise, and the tors (1, 11,24) have observed a decrease in the maximum medial layer was separated from the adventitial as deforce-generating ability of aorta and other conduit ves- scribed by Wolinsky and Daly (28). The length and wet sels. Because it has been demonstrated in many cases weight of the media were determined and the crossthat aortas from hypertensive animals have an increase sectional area calculated as wet weight/(density X in wall thickness that is due in part to an increase in the length) assuming a density of 1.05 g/cm3 (13). The aortas content of collagen and elastin (2, 15, 18), and because from two to three rats of similar age, body weight, and H34
0363-6135/79/0000-0000$01.25
Copyright
0 1979 the American
Physiological
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Society
ACTOMYOSIN
AND
HYPERTENSIVE
H35
AORTA
systolic blood pressure were cut in half lengthwise, and one-half of each was combined for actomyosin or DNA determination. Actomyosin was extracted as described previously (23). Briefly, 20-60 mg of media were homogenized in 1 ml 25 mM sodium phosphate (pH 7), I% /I-mercaptoethanol (BME) at 4OC. The homogenate was allowed to stand at 4OC for 2 h, and then sufficient sodium dodecylsulfate (SDS) was added to give a final concentration of 1% and the homogenate was incubated for 15 min at 37OC. After overnight storage at 4”C, the tissue debris was pelleted by centrifugation at 2500 g for 10 min at room temperature. A 2009.J aliquot of the supernatant was removed and frozen at - ,20°c to be used later for gel electrophoresis. A second aliquot of the supernatant was used for protein determination by the micro-Kjeldahl method. The pellet of insoluble material was dissolved in 2 ml of 1 M sodium hydroxide and its protein content was determined by the micro-Kjeldahl method. Gel electrophoresis. At the time of electrophoresis, 40 ~1 of a glycerol-tracking dye mixture (1 ml glycerol + 0.25 ml 5% pyronin Y in water) was added to the 200 ,ul aliquot of the supernatant obtained as described above. A sufficient volume of this solution to give 35-50 ,ug of supernatant protein and various volumes of purified skeletal muscle actin and myosin heavy chains to give l-10 pg of protein were placed in duplicate in the wells of a 0.1% SDS-10% acrylamide slab gel prepared according to the procedure of Laemmli (16) and Porzio and Pearson (20) with some modification (23). Electrophoresis was conducted for 5-6 h at 40 mA (0.13 mA/mm2). The slabs were stained for 30 min in 0.25% Coomassie blue, 45% methanol, and 10% glacial acetic acid in water and destained in 7.5% glacial acetic acid and 25% methanol for 14-16 h by diffusion. The gel slab was cut into strips corresponding to each well and scanned at 560 nm at a scanning rate of 2 cm/ min. The base line for each scan was determined by drawing a horizontal line through that portion of the scan corresponding to an area devoid of protein (lo), and the lateral limits of the peaks were defined by perpendicular lines drawn through minima in the scan. The area under the peak corresponding to actin or myosin was determined by planimetry. The amount of actin and myosin in the a&tic samples was calculated using the relationship between the amount of applied protein and the band density obtained from gels containing isolated skeletal muscle actin and myosin heavy chains as previously described by this laboratory (23). The amounts of actin, myosin heavy chain, and actomyosin (myosin heavy chains plus actin) in tissue samples were normalized with respect to tissue weight, total protein content (mount of protein in the homogenate pellet plus that in the supernatant), and DNA content. Purified skeletal muscle actin was prepared from acetone-dried powder of rabbit back muscle according to the method of Spudich and Watt (25), dialyzed against 1% SDS-l% BME overnight at room temperature, and stored in small aliquots at -2OOC. Rabbit skeletal muscle myosin was prepared according to the method of Nauss et al. (17), the light chains removed by the method of Perrie and Perry (19), and the purified myosin heavy
chains treated with 1% SDS-l% BME as described for purified actin. Small aliquots were stored at -2OOC. For each standard curve, fresh aliquots of actin-SDS and myosin heavy chain-SDS were used and mixed with glycerol-tracking dye in the same proportions as described for the tissue sample. The protein content of these skeletal muscle extracts was determined by the micro-Kjeldahl method and the purity was determined by gel electrophoresis. - As described previously (23), this method of extracting actomyosin from small samples of ar terial tissue results in nearly comple te extraction of the contractile protein (90%) and complete recovery of purified skeletal muscle actin or myosin. In addition, it has been shown (8, 23) that the density of Coomassie blue staining is directly related to the amount of protein applied to the gel over a range of l-10 pg and that there is a linear relationship between the amount of tissue applied to the gel and the area of the actin or myosin peak. DNA determination. DNA was determined on intimalmedial samples obtained as described above by the method of Ceriotti (4) as modified by Hubbard et al. (14). Commercially available (Sigma) DNA was used as a standard, and the concentration in the stock solution was determined by measuring its optical density at 260 nm (14). All tissue samples were determined in duplicate and the results were normalized to tissue mass or protein content I*Becau se it has been de term ined that the smo 0th muscle cell is the only cell in the rat aorta media (5) ? DNA content is a measure of the number of muscle cells, assuming normal diploid nuclei. From this measurement and assuming 6.2 pg DNA per mammalian cell (27)) the number of cells in the media was calculated. Statistically significant differences between groups was assessed by Student’s t test with P 5 0.05 as the criterion of significance. All statistical . calculations were performed data management with the assistance of the CLINFO system. RESULTS
Table 1 describes the physical characteristics of the animals and the aortic media segments. The body weight of both the WKY and SHR rats increased with animal age, with the body weight of the SHR increasing less rapidly than that of the WKY. Except at 3 wk of age, the systolic blood pressure of SHR rats was consistently elevated in all animals and, on the average, was significantly greater than that of the WKY. The systolic pressure of both groups tended to increase with age, with the larger increases occurring in the SHR group. The media weight, length, and calculated cross-sectional area tended to increase with animal age in both SHR and WKY groups, and a significant difference between groups in weight and area occurred after 6 wk of age. These increases in media weight and area in the SHR occurred later than the development of hypertension (i.e., a systolic pressure X50 mmHg). The absolute amount of total protein in the media segment increases significantly with increasing animal age in both the WKY and SHR groups (Table 2); however, only at 12 and 23 wk of age is the total protein
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H36 TABLE
C. L. SEIDEL
1. Physical
characteristics
Body Age, wk
of media from WKY
Wt,
Blood
g WKY
3
SHR
WKY
SHR
WKY
25 t 0.3
0.58 t 0.01
0.61 t 0.01
118 -+ 5*
152 t 4*j-
20 Ifr 0.3*
20 Ik 0.4*
35 t 0.6*
34 t 0.3*
0.54 If;: 0.01
0.58 t 0.02
268 t, 5*-f (15)
114 AZ 5
166 t 4-f
26 rt 0.6*,
30 t 0.4*t
40 zk 0.5*
40 t 0.3*
0.63 k O.Ol*
0.71 k 0.01*-t
341 k 3* (24)
128 t 2*
189 t, 3*t
29 t, 0.5*
36 -+ 0.7*-t
41 t 0.4
43 Ifr 0.4-f
0.67 t 0.01”
0.81 t O.Ol*t
152 zk 2*t
12
293 t 4* (15)
23
346 t 6*
All values same age.
TABLE
Age, wk
3
6
rt SE with
2. Total protein
number
of animals
in parentheses.
and DNA
contents
of media from Wistar,
TP/Media, mg
TP/Wet w/g
Wistar
WKY
SHR
2.09 t 0.2 (5)
2.8 t 0.1 (4)
2.8 -+ 0.2 (4)
208 t 21
4.75 t 0.5* (7)
4.0 + 0.2* (4)
4.2 t 0.1 (4)
239 t 14
6.4 +. 0.2*
7.5 t 0.2*-f
(8) 16
Wistar
* P < 0.05 relative
Wt,
age.
t P < 0.05 relative
SHR
174 t 10 175 t 6
at
DNA/Wet mgk
Wt,
Wistar
WKY
SHR
Wistar
WKY
SHR
35.2 t 4
35 t 2 (4)
34 t 2 (4)
3.62 t 0.21t
2.2 t 0.05
2.0 t 0.2
40 t 1 (4)
38 t, 1 (4)
4.36 t 0.53t
2.0 t 0.03
1.9 k 0.02
57 t 3* (7)
64 t 2* (7)
2.2 t 0.09
2.2 t 0.05
1.8 t 0.1
1.9 t 0.1
(6) 206 t, 11 209 -4 11 97.1 t 19*-f
(6) 254 t 5*
260 -4 13
to WKY
and SHR rats
DNA/Media, l-43
WKY
242 t 5”
to previous
WKY,
03)
8.4 t_ 0.5*-t (9)
23
SHR
(8)
are means
12
SHR
Media Area, mm’
26 t 0.5
161 -+ 2* (8)
WKY
Length, mm
16 t 0.5
(12)
SHR
Media
16 t 0.6
93 t 3
cm
Media Wt, mg
lOOt4
66 t, 1
WKY
71 + 3 (11)
6
Pressure, mmHg
and SHR rats
96.4 t 7-f
3.12 t 0.23-t
(16) 7.1 t 0.2*
9.0 + 0.2*-t
(12)
244 t 5
248 -+ 3
54 -L: 4 (11)
(12)
9.52 t 0.3t 202 t 4* (5) All values are means t SE with number of observations (23) and are included here for comparison purposes only.
69 t 5-t
(12)
43
TABLE
3. Actomyosin AM/Media,
Age, wk
content
Wistar
of media from
pg
in parentheses. TP, total protein. Data * P < 0.05 relative to previous age.
Wistar,
AM/Wet
WKY,
and SHR rats
Wt, mg/g
AM/R
WKY
SHR
Wistar
WKY
SHR
Wistar
WKY
SHR
49 t 9
38 t, 5
28 t 5
2.8 + 0.8
2.5 t 0.4
2.4 t 0.4
35 t 6
49 t 8”
1.7 t 0.6*
2.1 t 0.3
1.6 t 0.2
94 t 6*
93 t 9*
1.3 t 0.1
1.6 t 0.4
1.6 t 0.5
1.3 t 0.2
83 t 16 (4)
9.5 -4 0.4
6.5 t 0.4
5.0 t 0.9
6
615 t 76*t (7)
140 t 19 (4)
206 -+ 30” (4)
31.2 t 4.4*-t
7.1 t 1.0
10.1 t 1.3*
592 k 29*
702 t 63* (7)
22.5 t 1.2*
“23.8 t, 2.5”
(8) 926 t 62*t (9)
28.6 t 2-t
510 + 62
(12) 43
1,645 t, 93*t (5)
mghg
Wistar
107 t, 11 (4)
23
NM
SHR
97 t 8 (5)
16
w/g
WKY
3
12
on Wistar rats are from previous publication t P < 0.05 relative to WKY at closest age.
709 -+ 54-f
136 t, 2O*t
113 t 9-j-
17.3 t, 2.0
19.4 t 1.4
3.7 t 0.5-t
72 t 9
72 + 5
(12) 34.8 t 1.7f
All values are means t SE with number of observations in parentheses. Data on Wistar rats are from previous publication (23) and are included age. t P < 0.05 relative to WKY at closest age.
176 t 6-t AM, actomyosin; TP, total protein; here for comparison purposes only.
1.2 t 0.1* A, actin; M, myosin heavy chains. * P < 0.05 relative to previous
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ACTOMYOSIN.
AND
HYPERTENSIVE
AORTA
H37
content significantly greater in the SHR. When the total protein content is ‘expressed relative to the tissue wet weight, it tends to increase with animal age, but only significantly between 6 and 12 wk of age, and at no age is there a significant difference between the WKY and SHR groups. The absolute amount of DNA in the media segment also tends to increase with animal age; however, only between 6 and 12 wk of age is a significant increase observed. The DNA content of aortic media from SHR rats is greater than that from WKY rats only at 23 wk of age. There are no consistent changes with age or between groups in DNA content when expressed relative to tissue wet weight. The absolute amount of actomyosin in media from WKY and SHR rats tends to increase with animal age, reaching a stable level by 12 wk (Table 3). Only at 23 wk is the absolute amount of actomyosin in the media from SHR rats significantly greater than that from the WKY rat. When actomyosin is expressed relative to tissue wet weight or total protein content, it tends to increase with animal age; however, at any age there are no differences between the two groups. In both groups of animals, the actin-to-myosin weight ratio tends to decrease with animal age; however, the ratio is not statistically different even between 3 and 23 wk of age, and at any age there is no difference between the WKY and SHR groups. To assessintraspecies differences between aortas from Wistar, WKY, and SHR rats, various biochemical characteristics of aortas from WKY and SHR rats were compared to previously obtained values from Wistar rats (23). The DNA and actomyosin contents (Tables 2 and 3) of media from SHR and WKY rats is less than that from Wistar rats at all ages except 3 wk. When actomyosin is expressed relative to the number of cells as calculated from DNA (Fig. I), the actomyosin content of aorta from Wistar, WKY, and SHR rats is similar at the various ages studied.
01 0
I 3
I I 6 7
I 12
I 16
1 23
Rat Age (weeks) of amount of actomyosin per smooth muscle cell in thoracic aorta media from Wistar, WKY, and SHR rats as a function of age. All values are means t SE with number of observations in parentheses. Number of observations for WKY and SHR rats is same as in Table 3. Data on Wistar rats are from previous publication (23) and are included here for comparison purposes only. *P < 0.05 relative to previous age. FIG.
1. Comparison
DISCUSSION
Many investigators have observed that the maximum contractile response of in vitro preparations of large conduit arteries obtained from hypertensive animals is less than the response obtained from similar preparations from normotensive animals. This reduction in maximum force generation is observed in a variety of hypertensive models (1, 11, 24) is not unique to a given pharmacologic agent (1, 11, 24), is not due to a change in the lengthactive tension relationship (1, II), nor to the procedure used to prepare the in vitro preparation (12). These observations suggest that the reduced force-generating ability may be due to an alteration within the vascular smooth muscle cell. The particular alteration examined in this study was the actomyosin content of the smooth muscle cell. The present observations (Table 1) indicate that the weight and calculated cross-sectional area of media from SHR rats increase more than these parameters for media from WKY rats over the same age period even though total body development, as reflected in body weight, is the same or slower in the SHR. This apparent hypertrophy of SHR media is due to an increase in tot*al protein and water contents (Table 2) because the ratio of total protein to tissue wet weight remains constant between the two groups of animals at all ages. The three major proteins that contribute to the total protein value are collagen, elastin, and actomyosin. The present observations (Table 3) suggest that actomyosin is produced in a constant proportion to the sum of collagen and elastin because the ratio of actomyosin to total protein remains constant between the two groups at all ages. Normalization of actomyosin content to the number of cells in the media (Fig. 1) indicates that at all ages studied the amount of actomyosin per smooth muscle cell increases similarly with age in media from both SHR and WKY rats. These observations suggest the following with regard to the characteristics of the smooth muscle cells in the media from WKY and SHR rats. The first is that the maturation of the thoracic aorta with regard to its cellular actomyosin content occurs well after birth (between 6 and 12 wk, Fig. 1) and at similar times in both WKY and SHR rats. This increase in actomyosin content with maturation explains in part the observation of Shibata et al. (24) that aortas from 12-wk-old WKY and SHR rats develop more force than aortas from 4-wk-old rats. This relationship between cellular actomyosin content and maximum force development is similar to that observed in aortas from maturing Wistar rats (23). The second is that the media from SHR rats undergoes hyperplasia, but each cell contains the same amount of actomyosin as cells in media from WKY rats. The absolute amounts of DNA, total protein (Table 2), and actomyosin (Table 3) attain higher levels in media from SHR rats during maturation; however, they are produced in relative amounts similar to those observed in media from WKY rats. The explanation for this greater net production of cells and protein by the SHR aorta is unknown. The third is that the reported (11,24) lower maximum force-generating ability of aorta from SHR rats relative
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H38
C. L. SEIDEL
to aorta from WKY rats at a given age is not due to a reduction in actomyosin content. The amount of actomyosin per gram of tissue (Table 3) or per cell (calculated from DNA, Fig. 1) and the weight ratio of actin to myosin (Table 3) are not different at any age between the two
groups of animals. This implies that a mechanism other than a quantitative change in actin or myosin is responsible for-the smaller force-generating ability of aorta from SHR rats. Such a mechanism may involve a reorganization of the contractile filaments within the muscle cell (9) or the synthesis of an isoenzyme of myosin (21) Altern .atively, the red .uction in force genera .tion may not involve the contractile proteins at all, but some other aspect of the contractile process such as metabolism (7, 29), Ca availability (3, 26), or even the physical characteristics of the vessel wall. Several investigators (11, 24) have suggested that the reduction in maximum force development observed in conduit arteries is not due to the elevated systolic blood pressure. The present observations indicate that the increase in the absolute amount of actomyosin seen during maturation of the SHR media may also not be due to changes in systolic blood pressure. Table 3 indicates that only at 23 wk of age is the-absolute amount of actomyosin in SHR media significantly greater than in media from WKY rats, even though the systolic blood pressure (Table 1) had been significantly greater in the .SHR for at least the previous 18 wk. Second, the systolic blood pressure of the SHR rat increases significantly between 12 and 23 wk, yet the amount of actomyosin per media segment does -not change. Third, the 1.argest percent change in actomyosin per m.edia segment in both groups occurs between 6 and 12 wk of age, a time when no significant change in systolic blood pressure occurs in either group. Finally, with use of a multiple linear regression program of the CLINFO data management system, the correlation coefficient between actomyosin per media segment or per wet weight and blood pressure- taking into account the effect of age-was determined for both groups of animals between 3 and 12 wk of age and between 3 and 23 wk of age. The correlation coefficients (r c 0,431, P > 0.07) indicated that for both the WKY and the SHR groups, there was not a positive correlation between actomyosin content and systolic blood pressure. However, data in Tables 2 and 3 suggest that the number of cells (based on DNA content) and absolute amount of actomyosin in media from SHR rats attain final levels that are higher than those observed in media from WKY rats. This suggests that either the maturation process in the aortas of SHR rats proceeds at a more rapid rate than in aortas of WKY rats or that some other factor may contribute to differences observed between the two groups of animals. The nossibilitv that the aortic media from SHR rats mature&at a different rate than the media from WKY rats
raises the question of what is the appropriate control animal for studies designed to identify the cause of spontaneous hypertension.
This is especially important
in
light of the reported differences in force-generating ability of aorta from Wistar and WKY rats (6). To determine whether there is a biochemical rationale for selecting one type of normotensive control rat over another, previously published values for the actomyosin content of aorta from Wistar rats (23) were compared with present observations obtained from WKY and SHR rats.
This comparison indicates that at all ages beyond 3 wk, the amount of actomyosin per media wet weight or total protein is greater in aortas from Wistar rats and that it attains its “adult” level more rapidly (Tables 2 and 3). This greater actomyosin content is due to the presence of more cells (Table 3), with each cell containing the same amount of actomyosin as in aortas from WKY
and SHR rats (Fig. 1). The smaller ratio of actomyosin to total protein (Table 3) in the WKY and SHR aorta suggests that the smooth muscle cells in these media
produce a greater proportion of collagen and elastin than do the cells in the media from Wistar rats. These differences between the aorta from Wistar rats on one hand and WKY and SHR rats on the other, provide biochemical evidence favoring the use of the WKY rat as the control for the SHR. In addition, the greater amount of actomyosin per media weight of Wistar aortas provides a possible explanation
for the observation
of Cline-
Schmidt et al. (6) that aortas from Wistar rats develop more maximum force than aortas from WKY or SHR rats. In conclusion, these data suggest that the reduction in maximum force generation observed in in vitro preparations of aortas from spontaneously hypertensive rats is not due to a reduction in their actomyosin content. Either there is another alteration in actomyosin or some other explanation exists for the observed reduction in maximum force-generating ability. Second, there are several quantitative
biochemical differences between aortas from
WKY and Wistar rats, suggesting that the WKY rat may be a better control for studies involving the SHR rat. The author wishes to make special mention of the valued criticisms and suggestions made by Dr. J. C. Allen and Dr. Mark L. Entman during the performance of this work and the preparation of the manuscript and thanks Loris F. Rarrett for her extensive secretarial assistance. This work was supported in part by a grant from the American Heart Association (Texas Affiliate) and by National Institutes of Health Grant HL-17269 P-6 (Development Section of the National Heart and Blood Vessel Demonstration Center, Baylor College of Medicine, a grant-supported research project of the National Heart, Lung, and Blood Institute). Computational assistance was provided by the CLINFO Project, funded by the Division of Research Resources of the NIH under Grant RR-00350. Received 16 November 1978; accepted in final form 8 March 1979.
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H39
AORTA 18. OOSHIMA, A., UNDENFREEND.
G. C. FELLER, G. J. CARDINALE, S. SPECTOR, AND S. Increased collagen synthesis in blood vessels of hypertensive rats and its reversal by antihypertensive agents. Proc. 1974. NatZ. Acad. Sci. USA 71: 3019-3023, 19. PERRIE, W. T., AND S. V. PERRY. An electrophoretic study of the low-molecular-weight components of myosin. Biochem. J. 119: 3138, 1970. 20. PORZIO,
M. A., AND A. M. PEARSON. Improved resolution of myofibrillar proteins with sodium dodecylsulfate-polyacrylamide gel electrophoresis. Biochim. Biophys. Acta 490: 27-34, 1977. 21. Ross, R., AND S. J. KLEBANOFF. The smooth muscle cell. I. In vivo synthesis of connective tissue proteins. J. CeZZBioZ. 50: 159-171, 1971. 22. ROSZOWSKI,
R. R., J. D. WELTY, AND M. B. PETERSON. The amino acid composition of actin and myosin and Ca-activated myosin adenosine triphosphatase in chronic canine congestive heart failure. Circ. Res. 40: 191-198, 1977. 23. SEIDEL, C. L., AND R. A. MURPHY. Changes in rat aortic actomyosin content with maturation. Blood Vessels. 16: 98-fO8, 1979. 24. SHIBATA, S., K. KURAHASHI, AND M. KUCHII. A possible etiology of contractility impairment of vascular smooth muscle from spontaneously hypertensive rats. J. PharmacoZ. Exp. Ther. 185: 406417, 1973. 25. SPUDICH,
J. A., AND S. WATT. The regulation of rabbit skeletal muscle contraction. J. BioZ. Chem. 246: 4866-4871, 1971. 26. WEBB, R. C., AND R. C. BHALLA. Altered calcium sequestration by subcellular fractions of vascular smooth muscle from spontaneously hypertensive rats. J. MoZ. Cell. CardioZ. 8: 651-659, 1976. 27. WINICH, M., AND A. NOBLE. Quantitative changes in DNA, RNA, and protein during prenatal and postnatal growth in the rat. Deu. BioZ. 12: 451-466,1965. 28.. WOLINSKY, H., AND M. M. DALY. The isolation of intima-media samples from arteries. Proc. Sot. Exp. BioZ. Med. 135: 364-368, 1970. 29. ZEMPLENYI,
T. Metabolic intermediates, enzymes, and lysosomal activity in aortas of spontaneously hypertensive rats. AtheroscZerosis 28: 233-246, 1977.
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(With
content of maturing hypertensive rats the Technical
Assistance
normal
of R. Bowers)
Department of Medicine, Section of Cardiovascular Sciences, Baylor College of Medicine, Houston, Texas 77030 SEIDEL, C. L. Aortic actomyosin content of maturing normal and spontaneously hypertensive rats. Am. J. Physiol. 237(l):
H34-H39, 1979 or Am. J. Physiol.: Heart Circ. Physiol. 6(l): H34-H39, 1979.-Observations reported in the literature suggest that the maximum contractile response of aorta from spontaneously hypertensive (SHR) rats is reduced compared to that of normotensive controls and that the maximum contractile response of aorta from Wistar-Kyoto (WKY) rats is less than that of aorta from Wistar rats. A possible explanation for these differences is that there are differences in the amount of actomyosin per smooth muscle cell. To test this hypothesis, actomyosin, total protein, and DNA contents of thoracic aorta from 3-, 6-, 12-, and 23-wk-old SHR and WKY rats were determined and compared to similar values previously published by this laboratory for Wistar rats. The absolute amount of actomyosin increased with increasing age in aorta from both WKY and SHR rats, but increased more rapidly in the SHR so that by 23 wk it was significantly greater than that in the WKY. This increase was not correlated with changes in systolic blood pressure during maturation. Sufficient hyperplasia (based on DNA) was observed in the aorta of SHR relative to WKY rats, so that the amount of actomyosin per smooth muscle cell was not different between these two groups at any age studied. Comparison of aortas from SHR, WKY, and Wistar rats indicated that Wistar aortas had more actomyosin and a greater number of cells (based on DNA) per gram of media than aortas from either SHR or WKY rats, but at any age, all three groups had the same amount of actomyosin per smooth muscle cell. These observations suggest that a) the lower maximum contractile response of aorta from SHR rats is not due to a reduction in the amount of actomyosin per smooth muscle cell, b) there are biochemical differences in aorta between subspecies of rats, and c) the WKY rat may be a better control for studies involving SHR rats because the biochemical characteristics of their aorta are similar.
the smooth muscle cell is responsible for the synthesis of collagen and elastin (Zl), it is possible that the synthetic activity of the smooth muscle cell shifts toward the synthesis of connective tissue and away from the synthesis of actomyosin. A reduction in the actomyosin content of the muscle cell could result in a reduction in maximum force-generating ability. One of the objectives of this study was to test this hypothesis by comparing the actomyosin content of thoracic aorta from normotensive and spontaneously hypertensive rats. Second, to determine if changes in cellular actomyosin content were associated with changes in blood pressure, aorta from hypertensive and prehypertensive rats were studied. Third, because it has been demonstrated that in vitro preparations of aorta from Wistar rats develop more maximum tension than similar preparations from WistarKyoto rats (6) raising the question of which animal is the best control for the SHR rat, the present results were compared to previously obtained values for aortas from Wistar rats (23). METHODS
Actomyosin extraction. Male WKY and SHR rats were obtained from Taconic Farms and were allowed to accommodate to their new surroundings for 3-5 days before their systolic blood pressures were determined. Four age groups were studied: 3, 6, 12, and 23 wk. At the time of the experiment, the 3-wk-old rats were anesthetized with ether, and a needle-tipped cannula (23-gauge) was inserted retrograde into the aorta. Pressure was measured with a Statham P23Db pressure transducer and displayed on a Grass model 7 polygraph. The ether was removed and the systolic pressure was noted at the first genetic hypertension; maturation; thoracic aorta; vascular sign of a tactile response. The animal was then immedismooth muscle ately killed by thoracotomy. The systolic blood pressure in all other age groups was determined by the indirect PREVIOUS OBSERVATIONS (23) SUggWt a positive COrrelatail-cuff method and the animals were killed by cervical tion between changes in maximum force-generating abil- dislocation. The thoracic aorta between the subclavian ity of rat aorta and the cellular content of actomyosin. In and celiac arteries was removed from each animal, various animal models of hypertension, many investiga- cleaned of adhering fat, and slit open lengthwise, and the tors (1, 11,24) have observed a decrease in the maximum medial layer was separated from the adventitial as deforce-generating ability of aorta and other conduit ves- scribed by Wolinsky and Daly (28). The length and wet sels. Because it has been demonstrated in many cases weight of the media were determined and the crossthat aortas from hypertensive animals have an increase sectional area calculated as wet weight/(density X in wall thickness that is due in part to an increase in the length) assuming a density of 1.05 g/cm3 (13). The aortas content of collagen and elastin (2, 15, 18), and because from two to three rats of similar age, body weight, and H34
0363-6135/79/0000-0000$01.25
Copyright
0 1979 the American
Physiological
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Society
ACTOMYOSIN
AND
HYPERTENSIVE
H35
AORTA
systolic blood pressure were cut in half lengthwise, and one-half of each was combined for actomyosin or DNA determination. Actomyosin was extracted as described previously (23). Briefly, 20-60 mg of media were homogenized in 1 ml 25 mM sodium phosphate (pH 7), I% /I-mercaptoethanol (BME) at 4OC. The homogenate was allowed to stand at 4OC for 2 h, and then sufficient sodium dodecylsulfate (SDS) was added to give a final concentration of 1% and the homogenate was incubated for 15 min at 37OC. After overnight storage at 4”C, the tissue debris was pelleted by centrifugation at 2500 g for 10 min at room temperature. A 2009.J aliquot of the supernatant was removed and frozen at - ,20°c to be used later for gel electrophoresis. A second aliquot of the supernatant was used for protein determination by the micro-Kjeldahl method. The pellet of insoluble material was dissolved in 2 ml of 1 M sodium hydroxide and its protein content was determined by the micro-Kjeldahl method. Gel electrophoresis. At the time of electrophoresis, 40 ~1 of a glycerol-tracking dye mixture (1 ml glycerol + 0.25 ml 5% pyronin Y in water) was added to the 200 ,ul aliquot of the supernatant obtained as described above. A sufficient volume of this solution to give 35-50 ,ug of supernatant protein and various volumes of purified skeletal muscle actin and myosin heavy chains to give l-10 pg of protein were placed in duplicate in the wells of a 0.1% SDS-10% acrylamide slab gel prepared according to the procedure of Laemmli (16) and Porzio and Pearson (20) with some modification (23). Electrophoresis was conducted for 5-6 h at 40 mA (0.13 mA/mm2). The slabs were stained for 30 min in 0.25% Coomassie blue, 45% methanol, and 10% glacial acetic acid in water and destained in 7.5% glacial acetic acid and 25% methanol for 14-16 h by diffusion. The gel slab was cut into strips corresponding to each well and scanned at 560 nm at a scanning rate of 2 cm/ min. The base line for each scan was determined by drawing a horizontal line through that portion of the scan corresponding to an area devoid of protein (lo), and the lateral limits of the peaks were defined by perpendicular lines drawn through minima in the scan. The area under the peak corresponding to actin or myosin was determined by planimetry. The amount of actin and myosin in the a&tic samples was calculated using the relationship between the amount of applied protein and the band density obtained from gels containing isolated skeletal muscle actin and myosin heavy chains as previously described by this laboratory (23). The amounts of actin, myosin heavy chain, and actomyosin (myosin heavy chains plus actin) in tissue samples were normalized with respect to tissue weight, total protein content (mount of protein in the homogenate pellet plus that in the supernatant), and DNA content. Purified skeletal muscle actin was prepared from acetone-dried powder of rabbit back muscle according to the method of Spudich and Watt (25), dialyzed against 1% SDS-l% BME overnight at room temperature, and stored in small aliquots at -2OOC. Rabbit skeletal muscle myosin was prepared according to the method of Nauss et al. (17), the light chains removed by the method of Perrie and Perry (19), and the purified myosin heavy
chains treated with 1% SDS-l% BME as described for purified actin. Small aliquots were stored at -2OOC. For each standard curve, fresh aliquots of actin-SDS and myosin heavy chain-SDS were used and mixed with glycerol-tracking dye in the same proportions as described for the tissue sample. The protein content of these skeletal muscle extracts was determined by the micro-Kjeldahl method and the purity was determined by gel electrophoresis. - As described previously (23), this method of extracting actomyosin from small samples of ar terial tissue results in nearly comple te extraction of the contractile protein (90%) and complete recovery of purified skeletal muscle actin or myosin. In addition, it has been shown (8, 23) that the density of Coomassie blue staining is directly related to the amount of protein applied to the gel over a range of l-10 pg and that there is a linear relationship between the amount of tissue applied to the gel and the area of the actin or myosin peak. DNA determination. DNA was determined on intimalmedial samples obtained as described above by the method of Ceriotti (4) as modified by Hubbard et al. (14). Commercially available (Sigma) DNA was used as a standard, and the concentration in the stock solution was determined by measuring its optical density at 260 nm (14). All tissue samples were determined in duplicate and the results were normalized to tissue mass or protein content I*Becau se it has been de term ined that the smo 0th muscle cell is the only cell in the rat aorta media (5) ? DNA content is a measure of the number of muscle cells, assuming normal diploid nuclei. From this measurement and assuming 6.2 pg DNA per mammalian cell (27)) the number of cells in the media was calculated. Statistically significant differences between groups was assessed by Student’s t test with P 5 0.05 as the criterion of significance. All statistical . calculations were performed data management with the assistance of the CLINFO system. RESULTS
Table 1 describes the physical characteristics of the animals and the aortic media segments. The body weight of both the WKY and SHR rats increased with animal age, with the body weight of the SHR increasing less rapidly than that of the WKY. Except at 3 wk of age, the systolic blood pressure of SHR rats was consistently elevated in all animals and, on the average, was significantly greater than that of the WKY. The systolic pressure of both groups tended to increase with age, with the larger increases occurring in the SHR group. The media weight, length, and calculated cross-sectional area tended to increase with animal age in both SHR and WKY groups, and a significant difference between groups in weight and area occurred after 6 wk of age. These increases in media weight and area in the SHR occurred later than the development of hypertension (i.e., a systolic pressure X50 mmHg). The absolute amount of total protein in the media segment increases significantly with increasing animal age in both the WKY and SHR groups (Table 2); however, only at 12 and 23 wk of age is the total protein
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H36 TABLE
C. L. SEIDEL
1. Physical
characteristics
Body Age, wk
of media from WKY
Wt,
Blood
g WKY
3
SHR
WKY
SHR
WKY
25 t 0.3
0.58 t 0.01
0.61 t 0.01
118 -+ 5*
152 t 4*j-
20 Ifr 0.3*
20 Ik 0.4*
35 t 0.6*
34 t 0.3*
0.54 If;: 0.01
0.58 t 0.02
268 t, 5*-f (15)
114 AZ 5
166 t 4-f
26 rt 0.6*,
30 t 0.4*t
40 zk 0.5*
40 t 0.3*
0.63 k O.Ol*
0.71 k 0.01*-t
341 k 3* (24)
128 t 2*
189 t, 3*t
29 t, 0.5*
36 -+ 0.7*-t
41 t 0.4
43 Ifr 0.4-f
0.67 t 0.01”
0.81 t O.Ol*t
152 zk 2*t
12
293 t 4* (15)
23
346 t 6*
All values same age.
TABLE
Age, wk
3
6
rt SE with
2. Total protein
number
of animals
in parentheses.
and DNA
contents
of media from Wistar,
TP/Media, mg
TP/Wet w/g
Wistar
WKY
SHR
2.09 t 0.2 (5)
2.8 t 0.1 (4)
2.8 -+ 0.2 (4)
208 t 21
4.75 t 0.5* (7)
4.0 + 0.2* (4)
4.2 t 0.1 (4)
239 t 14
6.4 +. 0.2*
7.5 t 0.2*-f
(8) 16
Wistar
* P < 0.05 relative
Wt,
age.
t P < 0.05 relative
SHR
174 t 10 175 t 6
at
DNA/Wet mgk
Wt,
Wistar
WKY
SHR
Wistar
WKY
SHR
35.2 t 4
35 t 2 (4)
34 t 2 (4)
3.62 t 0.21t
2.2 t 0.05
2.0 t 0.2
40 t 1 (4)
38 t, 1 (4)
4.36 t 0.53t
2.0 t 0.03
1.9 k 0.02
57 t 3* (7)
64 t 2* (7)
2.2 t 0.09
2.2 t 0.05
1.8 t 0.1
1.9 t 0.1
(6) 206 t, 11 209 -4 11 97.1 t 19*-f
(6) 254 t 5*
260 -4 13
to WKY
and SHR rats
DNA/Media, l-43
WKY
242 t 5”
to previous
WKY,
03)
8.4 t_ 0.5*-t (9)
23
SHR
(8)
are means
12
SHR
Media Area, mm’
26 t 0.5
161 -+ 2* (8)
WKY
Length, mm
16 t 0.5
(12)
SHR
Media
16 t 0.6
93 t 3
cm
Media Wt, mg
lOOt4
66 t, 1
WKY
71 + 3 (11)
6
Pressure, mmHg
and SHR rats
96.4 t 7-f
3.12 t 0.23-t
(16) 7.1 t 0.2*
9.0 + 0.2*-t
(12)
244 t 5
248 -+ 3
54 -L: 4 (11)
(12)
9.52 t 0.3t 202 t 4* (5) All values are means t SE with number of observations (23) and are included here for comparison purposes only.
69 t 5-t
(12)
43
TABLE
3. Actomyosin AM/Media,
Age, wk
content
Wistar
of media from
pg
in parentheses. TP, total protein. Data * P < 0.05 relative to previous age.
Wistar,
AM/Wet
WKY,
and SHR rats
Wt, mg/g
AM/R
WKY
SHR
Wistar
WKY
SHR
Wistar
WKY
SHR
49 t 9
38 t, 5
28 t 5
2.8 + 0.8
2.5 t 0.4
2.4 t 0.4
35 t 6
49 t 8”
1.7 t 0.6*
2.1 t 0.3
1.6 t 0.2
94 t 6*
93 t 9*
1.3 t 0.1
1.6 t 0.4
1.6 t 0.5
1.3 t 0.2
83 t 16 (4)
9.5 -4 0.4
6.5 t 0.4
5.0 t 0.9
6
615 t 76*t (7)
140 t 19 (4)
206 -+ 30” (4)
31.2 t 4.4*-t
7.1 t 1.0
10.1 t 1.3*
592 k 29*
702 t 63* (7)
22.5 t 1.2*
“23.8 t, 2.5”
(8) 926 t 62*t (9)
28.6 t 2-t
510 + 62
(12) 43
1,645 t, 93*t (5)
mghg
Wistar
107 t, 11 (4)
23
NM
SHR
97 t 8 (5)
16
w/g
WKY
3
12
on Wistar rats are from previous publication t P < 0.05 relative to WKY at closest age.
709 -+ 54-f
136 t, 2O*t
113 t 9-j-
17.3 t, 2.0
19.4 t 1.4
3.7 t 0.5-t
72 t 9
72 + 5
(12) 34.8 t 1.7f
All values are means t SE with number of observations in parentheses. Data on Wistar rats are from previous publication (23) and are included age. t P < 0.05 relative to WKY at closest age.
176 t 6-t AM, actomyosin; TP, total protein; here for comparison purposes only.
1.2 t 0.1* A, actin; M, myosin heavy chains. * P < 0.05 relative to previous
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ACTOMYOSIN.
AND
HYPERTENSIVE
AORTA
H37
content significantly greater in the SHR. When the total protein content is ‘expressed relative to the tissue wet weight, it tends to increase with animal age, but only significantly between 6 and 12 wk of age, and at no age is there a significant difference between the WKY and SHR groups. The absolute amount of DNA in the media segment also tends to increase with animal age; however, only between 6 and 12 wk of age is a significant increase observed. The DNA content of aortic media from SHR rats is greater than that from WKY rats only at 23 wk of age. There are no consistent changes with age or between groups in DNA content when expressed relative to tissue wet weight. The absolute amount of actomyosin in media from WKY and SHR rats tends to increase with animal age, reaching a stable level by 12 wk (Table 3). Only at 23 wk is the absolute amount of actomyosin in the media from SHR rats significantly greater than that from the WKY rat. When actomyosin is expressed relative to tissue wet weight or total protein content, it tends to increase with animal age; however, at any age there are no differences between the two groups. In both groups of animals, the actin-to-myosin weight ratio tends to decrease with animal age; however, the ratio is not statistically different even between 3 and 23 wk of age, and at any age there is no difference between the WKY and SHR groups. To assessintraspecies differences between aortas from Wistar, WKY, and SHR rats, various biochemical characteristics of aortas from WKY and SHR rats were compared to previously obtained values from Wistar rats (23). The DNA and actomyosin contents (Tables 2 and 3) of media from SHR and WKY rats is less than that from Wistar rats at all ages except 3 wk. When actomyosin is expressed relative to the number of cells as calculated from DNA (Fig. I), the actomyosin content of aorta from Wistar, WKY, and SHR rats is similar at the various ages studied.
01 0
I 3
I I 6 7
I 12
I 16
1 23
Rat Age (weeks) of amount of actomyosin per smooth muscle cell in thoracic aorta media from Wistar, WKY, and SHR rats as a function of age. All values are means t SE with number of observations in parentheses. Number of observations for WKY and SHR rats is same as in Table 3. Data on Wistar rats are from previous publication (23) and are included here for comparison purposes only. *P < 0.05 relative to previous age. FIG.
1. Comparison
DISCUSSION
Many investigators have observed that the maximum contractile response of in vitro preparations of large conduit arteries obtained from hypertensive animals is less than the response obtained from similar preparations from normotensive animals. This reduction in maximum force generation is observed in a variety of hypertensive models (1, 11, 24) is not unique to a given pharmacologic agent (1, 11, 24), is not due to a change in the lengthactive tension relationship (1, II), nor to the procedure used to prepare the in vitro preparation (12). These observations suggest that the reduced force-generating ability may be due to an alteration within the vascular smooth muscle cell. The particular alteration examined in this study was the actomyosin content of the smooth muscle cell. The present observations (Table 1) indicate that the weight and calculated cross-sectional area of media from SHR rats increase more than these parameters for media from WKY rats over the same age period even though total body development, as reflected in body weight, is the same or slower in the SHR. This apparent hypertrophy of SHR media is due to an increase in tot*al protein and water contents (Table 2) because the ratio of total protein to tissue wet weight remains constant between the two groups of animals at all ages. The three major proteins that contribute to the total protein value are collagen, elastin, and actomyosin. The present observations (Table 3) suggest that actomyosin is produced in a constant proportion to the sum of collagen and elastin because the ratio of actomyosin to total protein remains constant between the two groups at all ages. Normalization of actomyosin content to the number of cells in the media (Fig. 1) indicates that at all ages studied the amount of actomyosin per smooth muscle cell increases similarly with age in media from both SHR and WKY rats. These observations suggest the following with regard to the characteristics of the smooth muscle cells in the media from WKY and SHR rats. The first is that the maturation of the thoracic aorta with regard to its cellular actomyosin content occurs well after birth (between 6 and 12 wk, Fig. 1) and at similar times in both WKY and SHR rats. This increase in actomyosin content with maturation explains in part the observation of Shibata et al. (24) that aortas from 12-wk-old WKY and SHR rats develop more force than aortas from 4-wk-old rats. This relationship between cellular actomyosin content and maximum force development is similar to that observed in aortas from maturing Wistar rats (23). The second is that the media from SHR rats undergoes hyperplasia, but each cell contains the same amount of actomyosin as cells in media from WKY rats. The absolute amounts of DNA, total protein (Table 2), and actomyosin (Table 3) attain higher levels in media from SHR rats during maturation; however, they are produced in relative amounts similar to those observed in media from WKY rats. The explanation for this greater net production of cells and protein by the SHR aorta is unknown. The third is that the reported (11,24) lower maximum force-generating ability of aorta from SHR rats relative
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H38
C. L. SEIDEL
to aorta from WKY rats at a given age is not due to a reduction in actomyosin content. The amount of actomyosin per gram of tissue (Table 3) or per cell (calculated from DNA, Fig. 1) and the weight ratio of actin to myosin (Table 3) are not different at any age between the two
groups of animals. This implies that a mechanism other than a quantitative change in actin or myosin is responsible for-the smaller force-generating ability of aorta from SHR rats. Such a mechanism may involve a reorganization of the contractile filaments within the muscle cell (9) or the synthesis of an isoenzyme of myosin (21) Altern .atively, the red .uction in force genera .tion may not involve the contractile proteins at all, but some other aspect of the contractile process such as metabolism (7, 29), Ca availability (3, 26), or even the physical characteristics of the vessel wall. Several investigators (11, 24) have suggested that the reduction in maximum force development observed in conduit arteries is not due to the elevated systolic blood pressure. The present observations indicate that the increase in the absolute amount of actomyosin seen during maturation of the SHR media may also not be due to changes in systolic blood pressure. Table 3 indicates that only at 23 wk of age is the-absolute amount of actomyosin in SHR media significantly greater than in media from WKY rats, even though the systolic blood pressure (Table 1) had been significantly greater in the .SHR for at least the previous 18 wk. Second, the systolic blood pressure of the SHR rat increases significantly between 12 and 23 wk, yet the amount of actomyosin per media segment does -not change. Third, the 1.argest percent change in actomyosin per m.edia segment in both groups occurs between 6 and 12 wk of age, a time when no significant change in systolic blood pressure occurs in either group. Finally, with use of a multiple linear regression program of the CLINFO data management system, the correlation coefficient between actomyosin per media segment or per wet weight and blood pressure- taking into account the effect of age-was determined for both groups of animals between 3 and 12 wk of age and between 3 and 23 wk of age. The correlation coefficients (r c 0,431, P > 0.07) indicated that for both the WKY and the SHR groups, there was not a positive correlation between actomyosin content and systolic blood pressure. However, data in Tables 2 and 3 suggest that the number of cells (based on DNA content) and absolute amount of actomyosin in media from SHR rats attain final levels that are higher than those observed in media from WKY rats. This suggests that either the maturation process in the aortas of SHR rats proceeds at a more rapid rate than in aortas of WKY rats or that some other factor may contribute to differences observed between the two groups of animals. The nossibilitv that the aortic media from SHR rats mature&at a different rate than the media from WKY rats
raises the question of what is the appropriate control animal for studies designed to identify the cause of spontaneous hypertension.
This is especially important
in
light of the reported differences in force-generating ability of aorta from Wistar and WKY rats (6). To determine whether there is a biochemical rationale for selecting one type of normotensive control rat over another, previously published values for the actomyosin content of aorta from Wistar rats (23) were compared with present observations obtained from WKY and SHR rats.
This comparison indicates that at all ages beyond 3 wk, the amount of actomyosin per media wet weight or total protein is greater in aortas from Wistar rats and that it attains its “adult” level more rapidly (Tables 2 and 3). This greater actomyosin content is due to the presence of more cells (Table 3), with each cell containing the same amount of actomyosin as in aortas from WKY
and SHR rats (Fig. 1). The smaller ratio of actomyosin to total protein (Table 3) in the WKY and SHR aorta suggests that the smooth muscle cells in these media
produce a greater proportion of collagen and elastin than do the cells in the media from Wistar rats. These differences between the aorta from Wistar rats on one hand and WKY and SHR rats on the other, provide biochemical evidence favoring the use of the WKY rat as the control for the SHR. In addition, the greater amount of actomyosin per media weight of Wistar aortas provides a possible explanation
for the observation
of Cline-
Schmidt et al. (6) that aortas from Wistar rats develop more maximum force than aortas from WKY or SHR rats. In conclusion, these data suggest that the reduction in maximum force generation observed in in vitro preparations of aortas from spontaneously hypertensive rats is not due to a reduction in their actomyosin content. Either there is another alteration in actomyosin or some other explanation exists for the observed reduction in maximum force-generating ability. Second, there are several quantitative
biochemical differences between aortas from
WKY and Wistar rats, suggesting that the WKY rat may be a better control for studies involving the SHR rat. The author wishes to make special mention of the valued criticisms and suggestions made by Dr. J. C. Allen and Dr. Mark L. Entman during the performance of this work and the preparation of the manuscript and thanks Loris F. Rarrett for her extensive secretarial assistance. This work was supported in part by a grant from the American Heart Association (Texas Affiliate) and by National Institutes of Health Grant HL-17269 P-6 (Development Section of the National Heart and Blood Vessel Demonstration Center, Baylor College of Medicine, a grant-supported research project of the National Heart, Lung, and Blood Institute). Computational assistance was provided by the CLINFO Project, funded by the Division of Research Resources of the NIH under Grant RR-00350. Received 16 November 1978; accepted in final form 8 March 1979.
REFERENCES N. R., AND H. V. SPARKS, Contractile response of vascular smooth muscle of renal hypertensive rats. Am. J. Physiol. 219: 340-344, 1970. 2. BERRY, C. L., AND S. E. GREENWALD. Effects of hypertension on the static mechanical properties and chemical composition of the rat aorta. Cardiovasc. Res. 10: 437-451, 1976. 1. BANDICK,
M. D. Sodium ions, calcium ions, blood pressure regulation, and hypertension: a reassessment and a hypothesis. Am. J. Physiol. 232:Cl65-C173, 1977 or Am, J. Physiol,: CeZZPhysioZ. 1: Cl65-Cl73,1977. CERIOTTI, G. Determination of nucleic acids in animal tissues. J. BioZ. Chem. 214: 59-70, 1955.
3. BLAUSTEIN,
4.
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ACTOMYOSIN
AND HYPERTENSIVE
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