Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan 48109 - 0656
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ABSTRACT |
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The object of this theme is to offer new perspectives on the effect of aging on signal-transduction pathways associated with agonist-induced contraction of smooth muscle cells from the colon. Smooth muscle cells from old rats (32 mo old) exhibit limited cell length distribution and diminished contractility. The observed reduced contractile response may be due to the effect of aging on signal-transduction pathways, especially an inhibition of the tyrosine kinase-Src kinase pathway, a reduced activation of the PKC pathway, and a reduced association of contractile proteins [heat shock protein 27 (HSP27)-tropomyosin, HSP27-actin, actin-myosin]. Levels of HSP27 phosphorylation are also reduced compared with adult rats.
heat shock protein 27; actin; tropomyosin; myosin; phosphatidylinositol 3-kinase; pp60src kinase
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INTRODUCTION |
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AGING AFFECTS SMOOTH MUSCLE contraction in different tissues. A tremendous amount of work has been cited in the literature on the effect of aging on skeletal muscle or on the cardiovascular system, yet very little work has been done on gastrointestinal smooth muscle. There is considerable evidence that the incidence of certain problems, such as dysphagia and constipation, increases dramatically with age. The areas at greatest risk of developing aging-related dysfunction are the upper gastrointestinal (GI) tract, particularly the oropharynx and esophagus, and the distal tract (colon and rectum). The amplitude and duration of the peristaltic pressure wave in hypopharyngeal sphincter of elderly humans are increased, but the amplitude of the upper esophageal sphincter is decreased (18). A decrease in maximal force and lower maximal velocity of shortening is observed in longitudinal smooth muscle from urinary bladder of aging rats (12). In colonic smooth muscle cells from aging rats, a decrease in calcium and potassium channel currents affects the initiation of contraction (25). Significant effects of aging on gastric and colonic motility in rats include slow gastric emptying of liquids and decreased fecal pellet transit and production (19). The age-dependent changes are both in the cholinergic neurotransmission as well as the response of smooth muscle to acetylcholine.
In this theme review we have focused on the effect of aging on signal-transduction pathways and on the association of contractile proteins in rat colon smooth muscle cells. In smooth muscle cells, receptor activation of PKC results in activation of sphingomyelinase and a concomitant production of ceramide (6, 17). Ceramide induces a sustained contraction of smooth muscle cells through a pathway involving the activation of MAP kinase and also through the activation of a nonreceptor tyrosine kinase pathway [activation of pp60Src and phosphatidylinositol 3-kinase (PI3)-kinase] (21).
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CHANGES OF FIBER TYPE COMPOSITION DURING AGING |
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Most of the studies relating to this topic are from the cardiovascular system. Cardiovascular function is altered with age in men and women. Advancing age is a risk factor for the development of cardiovascular disease. With advancing age, vascular function and reactivity are impaired, resulting in increased stiffness, decreased distensibility and compliance, and wall thickening (10).
Smooth muscle cells from the colon of old rats (32 mo old) exhibit
limited cell-length distribution and diminished contractility. Figure
1A shows the percent decrease
in cell length in response to acetylcholine or ceramide (0.1 µM) in
adult smooth muscle cells (35.7 ± 2.9%, n = 3;
P < 0.01) compared with aging smooth muscle cells
(16.3 ± 2.6%, n = 3; P < 0.05).
Figure 1B displays the length distribution of smooth muscle
cells from the colons of rats. The distribution profile of cells from
adult rats distributed over a wider range than cells from aging rats.
In the unstimulated state, the average cell length in aging rats
(45.3 ± 5.4 µm, n = 150; P < 0.05) is shorter (~25%) than cells from adult rats (59.5 ± 6.0 µm, n = 150; P < 0.05). Smooth
muscle cells from adult rat (12 mo old) had a span of cell-length
distribution (37-70 µm) with 55% of the cells in the range of
40-60 µm and 44% longer than 60 µm. Aging rat (32 mo old)
smooth muscle cell length spanned 26-57 µm, with 68% longer
than 40 µm. The shorter cell length in aging colon smooth muscle
cells suggests a shorter sliding distance between the contractile
proteins in response to contraction, which could be translated into a
decrease in the shortening and reduced plasticity observed.
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TYROSINE KINASE PATHWAY |
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Signal-transduction pathways mediating contraction of smooth
muscle cells involve cascades of protein phosphorylation. The changes
of protein kinases in aging cells leading to aberrant protein
phosphorylation have been reported in other cell systems. An increased
activation of EGF-receptor tyrosine kinase by EGF and transforming
growth factor- in the colonic mucosa of aged rats has been
described. Increased expression of pp60cSrc was
reported in gastric mucosa of aged rats (9).
Ceramide activates a tyrosine kinase-Src kinase-PI3-kinase pathway in
smooth muscle cells from the rabbit colon (6), and PI3-kinase seems to be a preferential substrate for pp60Src
kinase. Figure 2 shows that Src kinase
was not activated in aging rats (4.4 ± 1.2% increase,
n = 3; P = 0.07) compared with adult rats (39.6 ± 4.6% increase, n = 3;
P < 0.001). Figure 2 also shows that ceramide
activated P85 subunit of PI3- kinase in adult rats (52.8 ± 5.7%
increase, n = 3; P < 0.001) but not in
aging rats (4.0 ± 0.6% increase, n = 3;
P = 0.12). P85 subunit is present in both type IA and
type IB PI3-kinase, but the amounts and ratio of these two types are
unknown. The catalytic subunits of PI3- kinase (P110 and P110
)
are activated in aged rats. Ceramide has been shown to activate type IA
(P110
) PI3-kinase but not type IB (P110
) PI3-kinase in rabbit
smooth muscle cells (21). Figure 2 shows that P110
of
PI3-kinase was activated in response to ceramide in aging rats
(16.5 ± 2.2% increase, n = 3; P < 0.05) but to a much lesser extent than in adult rats (41.5 ± 5.3% increase, n = 3; P < 0.005).
Similar data were observed for P110
subunit of PI3-kinase: adult
rats (63.9 ± 4.7% increase, n = 3;
P < 0.005) in adult rats vs. (30.1 ± 5.1%
increase, n = 3; P < 0.05) in aging rats. The data suggest that the "tyrosine kinase-Src kinase"
pathway was inhibited in smooth muscle cells of aging rats.
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In colonic smooth muscle cells, ceramide-induced contraction as well as ceramide-induced activation of Src kinase (6) is Ca2+ dependent. Intracellular Ca2+ levels play an important role in modulating contraction-relaxation coupling of muscle cells. Age-associated decrease in intracellular calcium level in aging colon smooth muscle cells has been previously reported. Several studies suggest that aging affects either the number and/or functional properties of calcium channels in neurons of rat brain (11) and in muscle cells of rat heart (15). Inhibition of Src-kinase in response to ceramide in aging colon smooth muscle cells is probably due to a lower level of intracellular Ca2+ resulting from either/both the defect of extracellular calcium influx and/or a defect in calcium release from intracellular calcium stores. This suggestion needs to be confirmed in GI smooth muscle.
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EFFECT OF AGING ON K CHANNELS |
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Another topic has seldom been investigated in GI smooth muscle, compared with the abundance of work in cardiovascular and corporal smooth muscle. In general, modifications in expression of ion channels that regulate cell excitability most likely contribute to an impaired cell function in older people.
One characteristic of aging coronary arteries is their enhanced contractile responses to endothelial vasoconstriction factors, which increase the risk of coronary vasospasm in older people. There is evidence that aging induces increased responses of rat coronary and mesenteric arteries to K+ (13). The increase in K+-induced contractions in aging animals suggests a change in K+-channel function or expression as age progresses. Coronary arteries possess several types of K+ conductances; the large-conductance, voltage-dependent, and Ca2+-activated K+ channel (MaxiK) is particularly abundant and plays a key role in regulating arterial tone. (22).
MaxiK channels are diminished in aging coronary arteries in rats and humans. A diminution in the numbers of MaxiK channels leads to a decrease in the normal tonic hyperpolarizing force provided by the activity of these channels in coronary arteries and thus may contribute to the increased risk of coronary spasm in older people. Under physiological conditions, MaxiK channels are tonically active, serving as a hyperpolarizing force that opposes contraction. Thus reduced expression of MaxiK channels in aged coronary arteries would lead to a decreased vasodilating capacity and increased risk of coronary spasm and myocardial ischemia in older people.
Similarly, in corporal smooth muscle, MaxiK channels also seem to diminish with age. The mechanism for this tissue-specific aging-related change in channel density is unknown (23). It would be interesting to determine whether sexual hormones that diminish during aging (7) trigger these changes and whether such changes are contributing factors in GI smooth muscle.
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EFFECT OF AGING ON REGULATION OF CA2+ IN COLONIC SMOOTH MUSCLE CELLS |
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The major mechanisms involved in smooth muscle contractions not associated with changes in membrane potential are the release of inositol 1,4,5-trisphosphate and the regulation of Ca2+ sensitivity. Both mechanisms are important for the initial and sustained contractile response in colonic smooth muscle.
As in striated muscle, the amount of intracellular free Ca2+ is the key to regulation of smooth muscle tone. In the smooth muscle cells, Ca2+ binds to calmodulin, which is in contrast to striated muscles, in which intracellular Ca2+ binds to the thin filament-associated protein troponin (3). The calcium-calmodulin complex activates myosin light chain kinase (MLCK) by association with the catalytic subunit of the enzyme. The active MLCK catalyzes the phosphorylation of the regulatory light chain subunits of myosin (MLC20). Phosphorylated MLC20 activates myosin ATPase, thus triggering cycling of the myosin heads (cross-bridges) along the actin filaments, resulting in contraction of the smooth muscle. A decrease in the intracellular level of Ca2+ induces a dissociation of the calcium-calmodulin MLCK complex, resulting in dephosphorylation of the MLC20 by MLC phosphatase and in relaxation of the smooth muscle.
An age-related impairment of intrinsic sarcoplasmic reticulum (SR) function, i.e., the rate of Ca2+ uptake and the fractional rate of SR filling, and a decrease in SR volume are the most probable factors underlying the decreased speed of contraction in old fast-twitch motor units (8). Uncoupling of sarcolemmal excitation and SR Ca2+ release is assumed as a major determinant of weakness and fatigue. With increasing age, an increase of the number of RYR1 ryanodine receptor uncoupled from dihydropyridine recepter has been found in rat (soleus and extensor digitorum longus muscle) and human (vastus lateralis muscle). Dihydropyridine receptor RYR1 uncoupling leads to a significant reduction in the amount of releasable Ca2+ in skeletal muscles from old animals and humans. Similar studies would yield valuable information in GI smooth muscle.
In smooth muscle, the force/Ca2+ ratio is variable and depends partly on specific activation mechanisms. The effect of calcium-sensitizing agonists are mediated by GTP-binding proteins that generate protein kinase C or arachidonic acid as second messengers. The major mechanism of Ca2+ sensitization of smooth muscle contraction is through inhibition of the smooth muscle myosin phosphatase, thus increasing MLC20 phosphorylation by basal level activity of MLCK. The resulting myosin phosphorylation and subsequent smooth muscle contraction therefore occurs without a change in sarcoplasmic Ca2+ concentration. Ca2+ sensitization by the RhoA/Rho kinase pathway contributes to the sustained phase of the agonist-induced contraction in smooth muscle.
Rho kinase is known to inhibit MLC phosphatase and to directly phosphorylate MLC, altogether resulting in a net increase in activated myosin and the promotion of muscle contraction. Rho kinase antagonist Y-27632 would be a good tool to investigate the effect of aging on the response of colonic smooth muscle to contractile agonists.
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EFFECT OF AGING ON AGONIST-INDUCED ACTIVATION OF PKC |
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PKC has been shown to be activated in different cell systems such
as in colon smooth muscle cells (5), vascular smooth muscle cells, atrial myocytes, neonatal rat hearts. On stimulation with
contractile agonists, PKC- translocates to the cell membrane in
adult rabbit colon smooth muscle cells (5) and
associates with translocated RhoA and heat shock protein (HSP27)
(1, 2). In old rats, (Fig.
3) the
-isoform of PKC is activated in
aging rats, whereas three isoforms of PKC (
,
, and
) are
activated in normal adult rats.
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A class of proteins termed receptors for activated C kinases (RACKs) has been described that bind PKC. A functional impairment in PKC by RACK1 deficiency in aged rat brain cortex has been repoted (14). There is a possibility that, in aging rats, the inhibition of PKC activation may be affected by the dysfunction of RACKs.
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EFFECT OF AGING ON "PKC-MAP" KINASE PATHWAY |
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We have previously shown that in smooth muscle cells, contractile agonists, activate MAP kinase (17), and ceramide-induced MAP kinase activation is parallel, yet downstream, of Src and PI3-kinase activation (6). Western immunoblotting, using a phosphospecific MAP kinase antibody, indicates that MAP kinase is phosphorylated to a lesser extent in aging rats (16.5 ± 1.9% increase, n = 3; P < 0.005) than in adult rats (38.1 ± 0.5% increase, n = 3; P < 0.005). The difference in the activation of the PKC isoform may result in less activation of MAP kinase in aged rats, which would be also due to the lack of activation of the "tyrosine-Src kinase" pathway.
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EFFECT OF AGING ON THE ASSOCIATION OF THE CONTRACTILE PROTEINS (HSP27-TROPOMYOSIN, HSP27-ACTIN, ACTIN-MYOSIN) AND ON HSP27 PHOSPHORYLATION |
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Changes in contractile response of smooth muscle could also be due
to changes in the amount of contractile proteins and their association
with one another. In the intimal cells of human coronary arteries, the
amount of desmin decreases from childhood to adulthood and disappears
in the elderly. In aortic smooth muscle cells of aging rats, the
percentages of myosin and desmin decreases compared with young rats,
whereas actin and vimentin remained unchanged, as well as an
age-related increase in the proportion of the -actin isoform. We
have identified a low-molecular weight heat shock protein, HSP27, which
may be a putative contractile protein mediating a PKC-dependent
contraction in rabbit colonic smooth muscle cells (5). An
interaction between HSP27 and actin, as well as an interaction between
HSP27 and contractile proteins such as myosin, tropomyosin, and
caldesmon, was suggested in colonic smooth muscle cells. HSP27 plays a
role in cytoskeletal reorganization by increasing its stability and
forms a complex with other contractile proteins such as actin, myosin,
caldesmon, and tropomyosin. Our recent study (1) suggests
an interaction among HSP27, tropomyosin, and actin. On contraction
induced by acetylcholine or ceramide (0.1 µM), the amount of actin
immunoprecipitated with HSP27 increased, suggesting a functional
relationship between the association of contractile proteins and
agonist-induced contraction.
The expression of heat shock proteins has been shown to be markedly reduced with age in both cultured cells and in vivo (4), suggesting a defective protective mechanism with aging. In intact animals, hsp70 gene expression induced by the stress of restraint is also reduced with aging in rat aorta, vena cava, and adrenal glands when 6- and 24-mo-old Fischer rats were compared (24).
Immunoprecipitation followed by Western immunoblotting was applied to
detect the associations among contractile proteins. Figure
4 shows that the amount of tropomyosin
immunoprecipitated with HSP27 antibody increased on agonist stimulation
in both groups of rats but much more in adult rats (32.1 ± 1.1%
increase, n = 3; P < 0.005) than in
aged rats (9.3 ± 1.6% increase, n = 3;
P < 0.05). A similar result was obtained with
immunoprecipitation of actin with HSP27. The amount of actin
immunoprecipitated with HSP27 antibody increased on agonist stimulation
in both groups of rats but much more in adult rats (31.1 ± 2.9%
increase, n = 3; P < 0.005) than in
aged rats (12.9 ± 0.8% increase, n = 3; P < 0.05). The amount of myosin immunoprecipitated
with actin antibody increased on stimulation in both groups of rats but
much more in adult rats (51.3 ± 1.2% increase, n = 3; P < 0.005) than in aged rats (27.9 ± 3.3%
increase, n = 3; P < 0.05). The
~50% decrease in actin-myosin association in aged and adult rats
correlates with the percent decrease in shortening, as shown in Fig.
1A. Ceramide and acetylcholine induce phosphorylation of
HSP27 in colonic smooth muscle cells (1). Ceramide-induced
phosphorylation of HSP27 was reduced more in old rats (15.3 ± 6.2% increase, n = 3; P < 0.05) than
in adult rat smooth muscle cells (142.5 ± 13.6% increase,
n = 3; P < 0.001; Fig.
5). This may be due to an inhibitory
effect of aging on the tyrosine kinase-Src kinase pathway and on the
"PKC pathway."
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SUMMARY |
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Changes in the upstream cascade leading to the association of contractile proteins would affect contractility. Scarce data are available as to the effect of aging on GI smooth muscle. We have proposed a model whereby the association of HSP27 with other contractile proteins is an integral mechanism of PKC-mediated smooth muscle contraction. Inhibition of MAP kinase activation in aged rats may result in a decrease in HSP27 phosphorylation. MAP kinase phosphorylates MAPKAPK2 kinase, which further phosphorylates heat shock protein (HSP27) (16, 20). Reduced phosphorylation of HSP27 affects the associations of contractile proteins, leading to less contractility in aged smooth muscle.
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ACKNOWLEDGEMENTS |
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We thank D. Thomas and E. McDaid for assistance with technical editing and figure preparation.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-57020.
Address for reprint requests and other correspondence: K. N. Bitar, Div. of Pediatric Gastroenterology, Univ. of Michigan Medical School, 1150 West Medical Center Dr., MSRB 1, Rm. A520, Ann Arbor, MI 48109-0656 (E-mail: bitar{at}umich.edu).
10.1152/ajpgi.00264.2002
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