Yonago Acta medica 2001;44:61–68

Effects of Aging on Contraction and Ca2+ Mobilization in SmoothMuscle Cells of the Rat Coronary Artery
Fumiyo Kitamura-Sasaka*†, Keigo Ueda* and Yasuaki Kawai**Second Department of Physiology and †First Department of Internal Medicine, Tottori
University Faculty of Medicine, Yonago 683-0826 Japan
The present study was undertaken to investigate agonist-induced contraction and Ca2+
mobilization in the smooth muscles of coronary arteries isolated from young (16 weeks)
and aged (more than 72 weeks) rats. Ring segments of the rat coronary anterior descending arteries without endothelium were loaded with 40 µmol/L fura-2 acetoxymethyl
ester and mounted in an organ bath. Isometric tension was recorded using a force
displacement transducer, and the percentage of phosphorylated myosin light chain(MLC20) was measured with Western blotting. Administration of vasoactive substancessuch as 5-hydroxytryptamine (5-HT), prostagrandin F2α, histamine, endothelin-1 andangiotensin II (A-II) caused dose-dependent contraction in the isolated coronary arteries, and the active tension (mN force/mg tissue weight) evoked by these agonists was
significantly greater in the young rats than in the aged ones. On the other hand, theincrease in cytoplasmic calcium ([Ca2+]i) induced by the maximum concentration of
the agonists was not significantly different between the 2 groups. The percentage ofphosphorylated MLC20 induced by a maximum concentration of 5-HT or A-II was
significantly greater in the young rats than in the aged ones. The number of nuclei permedial area was fewer in the aged rats than in the young ones. These results suggestthat the age-dependent decrease in the agonist-induced contraction of rat coronary
arteries is attributable to a reduction of smooth muscle density in the media and/or an
impairment in the signal transduction pathway of the smooth muscle cells. One possible
site for the impaired pathway is either downstream from the [Ca2+]i increase or in someCa2+ independent secondary mechanisms.
Key words: aging; calcium; coronary artery; myosin light chain; vascular smooth muscle

It is generally accepted that the rise and fall ofcytoplasmic free calcium ([Ca2+]i) initiate contraction and relaxation, respectively, in smoothmuscles. The calcium ion binds to calmodulin,
and the calcium-calmodulin complex activatesmyosin light chain kinase (MLCK), which in turnleads to the phosphorylation of serine at position 19 on the myosin light chain (MLC20) andsubsequent contraction of the muscles (Johnson,
1987; Sweeney et al., 1994). It is also known,
however, that [Ca2+]i is not necessarily proportional to the levels of MLC20 phosphorylationand developed tension. Thus, secondary mechanisms that can modify Ca2+ sensitivity by changing the activities of phosphorylating and dephosphorylating enzymes of the MLC20, havebeen reported (Somlyo and Somlyo , 1994). A
number of neurotransmitters, hormones, andautacoids increase [Ca2+]i and cause a contraction of vascular smooth muscles. Such agonists
can increase force in permeabilized muscles inwhich [Ca2+]i is maintained at a constant level,
indicating an increase in Ca 2+ sensitivity(Kitazawa et al., 1989; Nishimura et al., 1990).

Abbreviations: A-II, angiotensin II; AM, acetoxymetyl ester; DTT, dithiothreitol; ET-1, endothelin-1; His,
histamine; 5-HT, 5-hydroxytryptamine; MLC, myosin light chain; MLCK, MLC kinase; PAGE, polyacrylamide gel electrophoresis; PGF2α, prostagrandin F2α; PSS, physiological salt solution; TCA, trichloroacetic acid61

F. Kitamura-Sasaka et al.

Numerous studies concerning age-dependentchanges in the contraction (Tuttle et al., 1966;
Tschudi et al., 1995) and relaxation (Fleisch etal., 1970; Hayashi and Toda, 1978; Kawai andOhhashi, 1989) of vascular smooth muscles
have been reported. These changes may lead toischemic heart disease, which occurs more
often in older patients than in the young, if coronary arteries are involved. Little is known in
the literature, however, about age-dependentchanges in Ca2+ mobilization and MLC20 phosphorylation in the smooth muscles of the coronary artery. In the present study, agonistinduced changes in [Ca2+]i, MLC20 phosphorylation, and developed tension were examinedin the isolated coronary arteries excised fromyoung and aged rats. We found that the level of
MLC20 phosphorylation in the smooth musclesof coronary arteries stimulated with vasoactive
substances was lower in aged rats than in young
ones with no difference in [Ca2+]i.

Measurement of isometric tension
After each ring was loaded with fura-2 acetoxymetyl ester (AM), it was mounted horizontally
between 2 hooks in an organ bath filled with 5
mL PSS (pH 7.4) on the stage of an inverted
fluorescence microscope (TMD-300, Nikon,
Tokyo, Japan). The PSS was maintained at 37± 0.5˚C and bubbled with 100% O2. A hookholding one end of the ring was fixed to the wallof the bath, and another hook was connected to
the lever of a force displacement transducer(UL-10GR, Minebea, Nagano, Japan). The
isometric tension detected by the transducer
was amplified and recorded in a data analyzer(MacLab Mk III, AD Instrument, Castle Hill,
Australia). The specimen was allowed to equilibrate for 60 min at a resting tension of 1.8 mN.
The optimal range of resting tension for obtaining maximum contraction was 1.5 to 1.9 mN in
young rats and 1.6 to 2.2 mN in aged rats. Afterthe equilibration period, the rings were contracted by 80 mmol/L KCl solution; then thedose-response curves for 5-hydroxytryptamine(5-HT), prostagrandin F2α (PGF2α), histamine(His), endothelin-1 (ET-1 ) and angiotensin II(A-II) were obtained by adding each agonist
into the PSS for 15 to 20 min for ET-1 and 5 to
10 min for the rest of agonists. At the end ofeach experiment, the ring was removed fromthe organ bath, and the wet weight of the preparation was measured. The active tension produced by the agonists was normalized as force(mN) per wet tissue weight (mg).

Materials and Methods
Wistar male rats aged 16 weeks (young, n = 21)
and 72 weeks (aged, n = 21) were killed with
25% urethane (0.02 mL/g, intraperitoneally),
and the left anterior descending coronary arterywas excised from the heart and placed in normal
physiological salt solution (PSS). All procedures were reviewed and approved in accordance with the Guidelines for Animal Experimentation at Tottori University Faculty of Medicine, Yonago, Japan, and conformed to “Guiding
Principles for the Care and Use of Animals in
the Field of Physiological Sciences” publishedby the Physiological Society of Japan.

Measurement of [Ca2+]i
The arterial ring was loaded for 3 h at 37˚C withfluorescence dye 40 µmol/L fura-2 AM. Chremophor EL (0.02%) was added to improve the dyeloading. After loading, the tissue was washedout 2 or 3 times with the PSS. The measurement of fluorescence was performed using an
inverted fluorescence microscope equippedwith a fluorometric system (Quanti Cell 700,
Applied Imaging, Newcastle, United Kingdom).
Fura-2 was excited with the light of a 100 W
xenon lamp at wavelengths of 340 nm and 380

Tissue preparation
The isolated arterial segment was trimmed offat, connective tissue and myocardia, and was
cut into a ring segment of 4 mm long. The intimal surface of the segment was rubbed gently
with a single human hair to remove the endothelium. Removal of the endothelium was confirmed by the absence of relaxant response toacethylcholine.
62

Aging and coronary artery

nm. The fluorescence emission (F340 and F380)
was recorded at 510 nm and monitored with anintensified computer-controlled display camera. After the noise signal and autofluorescence
had been subtracted, the ratio (F340/F380) of
fluorescence was calculated as an indicator of[Ca2+]i.

agent. Densitometry of immunoblot and quantitation of absorbance peaks were performed
with a National Institutes of Health image
analysis (MD 20892) equipped with a recording
integrator. The extent of MLC20 phosphorylation is expressed as the percentage of MLC20 ineither the mono- or diphosphorylated form.

Measurement of MLC20 phosphorylation

Morphometric analysis

The extent of MLC20 phosphorylation in thearterial ring was measured by separation of
non-, mono- and diphosphorylated forms byglycerol-polyacrylamide gel electrophoresis(PAGE) followed by electrophoretic transfer ofthe proteins to a poly vinylidene di-fluoride
membrane (Persechini et al., 1986). The relative amount of each form was quantified by animmunoblot procedure as described elsewhere(Hathaway and Haeberle, 1983). After incubation with each agonist, arterial rings were frozen by immersion in acetone containing 10%
trichloroacetic acid (TCA) and 10 mmol/L dithiothreitol (DTT) cooled with dry ice. The frozen tissues were washed twice with acetone
containing 10 mmol/L DTT to remove the TCA
and then dried. The dried tissues were cut into
small pieces and homogenized for 2 min at 4˚C,using a homogenizer in 35 µL of glycerol-PAGE
sample buffer which contained 20 mmol/L Tris
base-22 mmol/L glycine (pH 8.6), 10 mmol/L
DTT, 8 mol/L urea and 0.1% bromphenol blue.
The urea-solubilized samples (12 µL) were subjected to glycerol-PAGE / immunoblot analysis, using the specific MLC20 antibody (Seto etal., 1990). The region containing MLC20 was
visualized as bands, using an enhanced chemiluminescence Western blotting detection re-

The isolated left anterior descending coronary
artery was gently flushed with 10% formaldehyde and fixed in the solution for at least 24 h.
After routine processing, the tissues were embedded in paraffin. They were cut and stainedwith hematoxylin and eosin or elastica van
Giesen stains. The thickness of the muscular
media was measured under a light microscopeat 4 different sites, and finally the average of the
4 sites was determined. The area of the media
was planimetrically quantified using the National Institutes of Health image analysis, andthe number of nuclei in the media was counted.

Solutions
The composition of the standard PSS was asfollows (mmol/L): 130.0 NaCl, 5.4 KCl, 2.5
CaCl2, 1.0 MgCl2, 5.5 glucose and 10.0 HEPES.
Substituting KCl for equimolar NaCl in the standard PSS made a high K+ solution (80 mmol/L).

Drugs and chemicals
The following compounds were used: fura-2
AM (Dojin, Kumamoto, Japan), A-II, cremophor EL, His, 5-HT, PGF2α, (Sigma, St. Louis,MO) and ET-1 (Peptide, Osaka, Japan). The
concentration of agonists was expressed as
the final organ bath concentration.

Table 1. Body weight, heart weight and coronary
ring weight
Body weight
(g)

Heart
Coronary ringweight (g) weight (mg)Young [16] 388.3 ± 8.3 1.3 ± 0.4
0.34 ± 0.01
Aged [16] 578.8 ± 10.8** 1.7 ± 0.5** 0.96 ± 0.03**
The data are given as mean ± SEM.
[ ], number of animals.
Significantly different from young rats: ** P < 0.01.

63

Statistics
The data were expressed as mean ± SEM(n), where n refers to the number of animals. Student’s t test was used to determine
the statistical difference; P < 0.05 was considered significant.

F. Kitamura-Sasaka et al.

100 µm

100 µmFig. 1. Left anterior descending coronary arteries isolated from a young rat (left) and an aged rat (right). The
media is thick and the vessel wall is dilated in the aged. Elastica van Giesen stain, original magnification × 65.

Table 2. Morphometric parameters of the coronary arteries

Results
Body weight, heart weight and coronary ringweight were significantly heavier in aged rats
than in young rats (Table 1). Figure 1 shows
cross-sections of the left anterior descending
coronary artery of young (left) and aged rats
(right). The medial thickness of the arteries was
53.0 ± 1.5 µm in young rats and 75.3 ± 2.4 µm
in aged rats, being significantly (P < 0.01) different between the 2 groups (Table 2). The
number of nuclei per medial area was significantly less in aged rats than in young ones
(Table 2).
Active tension (mN force/mg tissue weight)
induced by 80 mmol/L KCl was smaller in aged

[8]
2.5

Tension1.5(mN/mg tissue)

1.8

0.9

[8]
[5]
[8]

[8]
[8]

*

*

[5]

0.0
7

6

5

5-HT

4

3
(–log mol/L)

0.4
[8]

[8]

0.4
0.2

0.0

[8]

0.8
0.6

** **

[8]

0.5

1.0
[8]

[5]

[8]

0.6

1.2

[8]

[8]

0.8
0.7

1.4

[8]

Density of
nuclei(/103 µm2
medial area)

rats (0.72 ± 0.06) than in young rats (2.01 ±0.11). Figure 2 shows the dose-response curvesof contraction in isolated rings of the coronary
artery for 5-HT (left), PGF2α (middle) and His

1.6

[8]

1.0
0.5

[5]

[8]

Medialarea(104 µm2 )Young [16] 53.0 ± 1.5
3.3 ± 0.03 4.4 ± 0.4
Aged [16] 75.3 ± 2.4** 10.3 ± 0.6** 1.3 ± 0.1**
The data are given as mean ± SEM.
[ ], number of animals.
Significantly different from young rats: ** P < 0.01.

(mN/mg tissue)(mN/mg tissue)

2.0Medialthickness
(µm)

0.2

** ** **
** **
[8]

7

6

5

PGF2α

[8]

4

3
(–log mol/L)

0.1

[8]

[5]

[8]

[5]

[8]

[8]

[8]

0.3
[8]

**

** ** **

0.0
7

6

5

His

4
3
(–log mol/L)

Fig. 2. Dose-response curves of 5-hydroxytryptamine (5-HT, 10–7–10–3 mol/L), prostagrandin F2α (PGF2α,
10–7–10–3 mol/L) and histamine (His, 10–7–10–3 mol/L) in isolated rings of the coronary arteries of young( ) and aged ( ) rats. The contractile responses are expressed as active tension in mN/mg tissue andshown as mean ± SEM. Significantly different between groups (*P < 0.05, **P < 0.01). [n], number ofanimals.

64

Aging and coronary artery(mN/mg tissue)(mN/mg tissue)

3.5

1.2

[5]

[8]

3.0
2.5

Tension

[8]

[5]

[8]

[5]

**

**

7

6
(–log mol/L)

1.0
0.8
[8]

2.0

[8]
0.6

1.5

[8]
[8]

[8]

1.0

[8]

0.5

[8]
[8]

0.0
10

[8]

** **
9

8

ET-1

[5]

0.4
[8]

** **

0.2

[8]

0.0
7

6
(–log mol/L)

9

** **
8

A-II

Fig. 3. Dose-response curves of endothelin-1 (ET-1, 10–10–10–6 mol/L) and angiotensin II (A-II, 10–9–10–6
mol/L) in isolated rings of the left anterior coronary arteries of young ( ) and aged ( ) rats. The contractile responses are expressed as active tension in mN/mg tissue and shown as mean ± SEM. Significantly
different between groups (*P < 0.05, **P < 0.01). [n], number of animals.

(right). Active tension evoked by 10–6 mol/L to
10–3 mol/L of these agonists was significantly
greater in young rats (solid line) than in aged
rats (dotted line). ET-1 and A-II also caused
concentration-related contractions (Fig. 3) inthe coronary rings from young (solid line) andaged rats (dotted line). The active tension induced by 10–9 to 10–6 mol/L of these peptides
was greater in young rats than in aged rats. In
Table 3, the maximum contraction of the arteries induced by the agonists is compared between young and aged animals. It was 3 times
greater in the young group than in the aged
group when it was expressed as mN force per
mg tissue weight. On the other hand, when it
was normalized as percentage of 80 mmol/L

KCl-induced contraction, there was no significant difference between the 2 groups. The increase in [Ca2+]i induced by 80 mmol/L KCl
and maximum concentration of agonists wascomparable in the 2 groups (Fig. 4).
Table 4 shows the percentage of phosphorylated MLC20 (mono- and diphosphorylated)in rat coronary arteries. The percentage ofphosphorylated MLC20 during rest (control)
was significantly lower in the young group thanin the aged group. Addition of high K solution,
10–4 mol/L 5-HT, and 10–7 mol/L A-II increased
the percentage in both groups. The phosphorylation level during contraction was significantly higher in the young rats than in the aged
rats.

Table 3. Summary of agonist-induced contraction
Maximum active tension
Young
Aged
(mN/mg)(mN/mg)Percentage of KCl contraction
Young
Aged
(%)
(%)

5-HT (10–4 mol/L)
2.46 ± 0.11
0.96 ± 0.06*
122.4 ± 5.4
133.4 ± 7.8NS–4
PGF2α (10 mol/L)
1.56 ± 0.13
0.45 ± 0.09*
77.5 ± 6.3
62.2 ±12.2NS
His(10–4 mol/L)
0.76 ± 0.06
0.29 ± 0.02*
37.6 ± 3.1
40.1 ± 2.8NS–7ET-1 (10 mol/L)
2.96 ± 0.23
0.88 ± 0.04*
147.6 ±11.7
122.4 ± 6.0NS
A-II(10–7 mol/L)
1.04 ± 0.07
0.36 ± 0.03*
51.7 ± 3.7
49.5 ± 3.9NS
The data are given as mean ± SEM.
A-II, angiotensin II; ET, endothelin; His, histamine; 5-HT, 5-hydroxytryptamine; NS, not significant; PGF2α,
prostagrandin F2α.
Significantly different from young rats: *P < 0.05.

65Change in ratio (F340/F380)

F. Kitamura-Sasaka et al.

from aged rats compared with those from younger
rats (Table 2), which is consistent with previous
histological observation (Hüsken et al., 1997).
Thus, the smooth muscle fibers seem to be dispersed in the media of coronary arteries fromaged rats, and this dispersion is probably one of
the reasons for the decrease in agonist-induced
active tension. On the other hand, contractile
responses of rat coronary arteries to 5-HT
(Nyborg and Mikkelsen, 1988; Tschudi andLüscher, 1995), ET-1 (Tschudi and Lüscher,1995) and KCl (Tschudi and Lüscher, 1995) havebeen shown to increase with age. In thesestudies, however, the amount of response wasestimated as the developed force divided by thelength of ring segment. Thus, the increase inwall thickness of the vessel isolated from aged
animals was not taken into account.
Not only the dispersed arrangement ofsmooth muscle fibers but also a reduction incontractile response of the individual muscles
to agonists seems to take part in the agedependent decrease in active tension, because
the level of MLC20 phosphorylation in the coronary arteries stimulated with the agonists was
lower in the aged rats than in the young ones
(Table 4). Thus, it is important to know whichpart of the signal transduction for agonistinduced contraction is modified with age. The
function of certain receptors, such as the beta
adrenoceptor, decreases during the aging process (Fleisch et al., 1970; Hayashi and Toda,
1978). In the present experiment, however, thedecrease in active tension was observed with all

0.3

0High K 5-HT PGF2α His

ET-1

A-II

Fig. 4. Increases in ratio (F340/F380) induced by
maximal concentration of agonists in isolated ringsof the coronary arteries of young ( , n = 8) and aged
( , n = 8) rats. The data are expressed as mean ±SEM. Maximal concentration; 10–4 mol/L for 5hydrotryptamine (5-HT), prostagrandin F2α (PGF2α),and histamine (His) or 10–7 mol/L for endothelin-1(ET-1) and angiotensin II (A-II).

Discussion
The present results showed that the medial
thickness and cross-sectional area of rat coronary arteries increased with age, and the number of nuclei per medial area decreased withage. On the other hand, the active tension (mNforce/mg of tissue weight) induced by agonists
decreased with age. The decrease in active tension was associated with a reduction in theagonist-induced elevation of the phosphorylated MLC20 level in the tissues from aged animals,
although there was no significant difference in[Ca2+]i.
A number of studies have demonstrated agedependent changes in contractile and relaxant
responses of vascular smooth muscles to vasoactive agents. The sensitivity to norepinephrineof rat aortic strips was greatly reduced with age
when the contractile response was estimated as
milligram force per milligram wet weight(Tuttle, 1966; Cohen and Berkowitz, 1976),
which was the same method we used in thepresent study. The decreased sensitivity was
associated with an increased thickness of themedia and a coarsening of the smooth muscle
fibers (Tuttle, 1966). The number of nuclei permedial area decreased in the coronary arteries

Table 4. Percentage of phosphorylated MLC20
in the smooth muscle cells of the coronary
arteries
Young

Aged

Control[5] 19.9 ± 1.9 28.0 ± 2.6**
High K (80 mmol/L) [5] 63.5 ± 1.1 56.8 ± 2.2**
5-HT (10–4 mol/L) [5] 71.3 ± 1.5 66.3 ± 2.2**
A-II(10–7 mol/L) [5] 76.2 ± 4.1 58.2 ± 6.5**
The data are given as mean ± SEM.
[ ], number of animals.
A-II, angiotensin II; K, KCl solution; 5-HT, 5-hydroxytryptamine; MLC, myosin light chain.
Significantly different from young rats: **P < 0.01.

66Aging and coronary artery

agonists including 5-HT, PGF2α, His, ET-1 andA-II. Furthermore, the active tension of coronary arteries induced by 80 mmol/L KCl solution was also smaller in the aged rats than in theyoung ones. Hence, the age-dependent change
in rat coronary smooth muscles is likely to occur at a common pathway of the signal transduction distal to the receptor. This is also supported by the result that the agonist-induced increase in [Ca2+]i was not significantly different
between young and aged ones (Fig. 4). Taken
together, the age-dependent change in the signal transduction pathway may occur either
downstream from the [Ca2+]i increase, including the binding of Ca2+ to calmodulin and theactivation of MLCK by Ca-calmodulin complex, or in the secondary mechanism(s) which is(are) independent of [Ca2+]i.
The sensitivity of coronary arteries to Ca2+
has been investigated in pigs (Nyborg and
Mikkelsen, 1988; Noda et al., 1996) but not inrats. The present results show that 10–4 mol/L
5-HT and 10–7 mol/L ET-1 increased [Ca2+]i toalmost the same level as 80 mmol/L KCl did(Fig. 4), and caused a 120–150% contraction of
the KCl contracture (Table 3) in the rat coronary arteries. This is consistent with previous
results in porcine coronary arteries where agonists such as carbachol, 5-HT, PGF22α, endoperoxide analogue U-46619 and ET-1 produced
a greater force for a given [Ca2+]i compared tothat seen during KCl contracture (Bradley andMorgan, 1987; Kodama et al., 1994). These results suggest that a secondary mechanism(s)that can modify, independently of [Ca2+]i, thephosphorylation of MLC20 is (are) playing acertain role in the regulation of smooth muscle
contraction in the coronary arteries.
Studies using permeabilized smooth muscles revealed that a GTP-binding protein inhibits MLC20 phosphatase and increases Ca2+ sensitivity (Somlyo and Somlyo, 1994) and that therho p21 family is involved in the GTPγSenhanced Ca2+ sensitivity of smooth muscle
contraction (Ichi-Hirata et al., 1992; Gong et al,
1996; Noda et al., 1996). A recent study further
demonstrated that rho inhibits MLC phosphatase through activation of rho-kinase (Kimura etal., 1996). On the other hand, Eto and cowork-ers (Eto et al., 1995; Li et al., 1998) suggested
that CPI-17 protein accounts largely for thepathway between protein kinase C and MLC
phosphatase. Effects of aging on the functionof rho, rho-kinase and CPI-17 have not yet been
investigated in smooth muscle cells. Further
studies will be needed to clarify whether they
are involved in the age-dependent change in thecontractile response of coronary arteries tovasoactive substances.
In conclusion, the active tension (mN offorce/mg of tissue weight) of isolated rat coronary arteries induced by agonists decreased
with age, which was associated with the increase in the medial thickness and the reductionof the agonist-induced elevation of phosphorylated MLC20. The age-dependent change isattributable to a reduction of smooth muscle
density in the media and/or an impairment in
the signal transduction pathway in the smoothmuscle cells. A possible site for the impaired
pathway is either downstream from the [Ca2+]i
increase or in some Ca2+-independent secondary mechanism(s).

Acknowledgments: The authors thank Dr. Minoru
Seto, PhD, Life Science Center, Asahi Chemical Industry, Co. Ltd., Shizuoka 410-23 Japan for kindly
giving us the anti-MLC20 antibody and helping uswith fruitful discussion. The authors also thank Dr.
A. Ohtahara, First Department of Internal Medicine,
Tottori University Faculty of Medicine, for his technical assistance and fruitful discussion and Mr. Toshio
Kameie, Department of Anatomy, Tottori University
Faculty of Medicine, for his assistance in the histological experiments.

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Received January 12, 2001; accepted January 17, 2001
Corresponding author: Prof. Yasuaki Kawai

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