Full text of DOI:10.1093/gerona/55.11.B545

Journal of Gerontology: BIOLOGICAL SCIENCES
Copyright 2000 by The Gerontological Society of America
2
000, Vol. 55A, No. 11, B545–B551
Age-Related Decreases in Lymphocyte Protein
Kinase C Activity and Translocation Are
Reduced by Aerobic Fitness
Hoau-Yan Wang, Theodore R. Bashore, Zung Vu Tran, and Eitan Friedman1
1
2,3
4
1Department of Pharmacology and Physiology, MCP Hahnemann School of Medicine, Philadelphia, Pennsylvania.
2Department of Psychology, University of Northern Colorado, Greeley.
3Moss Rehabilitation Research Institute, Philadelphia, Pennsylvania.
4Department of Preventive Medicine and Biometrics, University of Colorado Health Sciences Center, Denver.
This study investigated the effects of advancing age and long-term aerobic fitness on lymphocyte protein kinase C
(PKC) activity and translocation. Lymphocytes were obtained from young (20–36 years old) and older (61–78
years old) healthy men who were either aerobically conditioned or deconditioned. Both baseline PKC activity and
the response of this enzyme to the direct PKC stimulating agent, phorbol 12-myristate, 13-acetate (PMA) or to the
mitogen, phytohaemagglutinin (PHA), were measured in partially purified extracts of cytosolic and membranous
fractions of lymphocytes. Basal PKC activity, PMA-induced redistribution of PKC, and PHA-induced enhance-
ment of PKC activity were reduced among older subjects in both lymphocyte cytosolic and membranous fractions.
However, the magnitudes of these reductions were smaller among the older subjects who were aerobically fit.
Lymphocyte PKC activity and translocation may be biological markers of aging, and the maintenance of aerobic
fitness into later life may serve to slow the rate at which activation of this enzyme declines during senescence.
ROTEIN kinase C (PKC) is a serine/threonine protein-
phosphorylating enzyme. It is widely distributed through-
tion may involve the activation of PKC (15). In this article,
we describe an extension of our work to include the effects
of aging and aerobic fitness on PKC activity and its translo-
cation in the immune system. To do so, we tested the effects
of two known mitogenic agents, phorbol 12-myristate, 13-
acetate (PMA) and phytohaemagglutinin (PHA), on lym-
phocytes obtained from young and older sedentary and aer-
obically fit males. PMA stimulates PKC activity directly,
inducing a translocation of PKC from cytosol to membrane,
thus redistributing intracellular PKC activity. The precise
mechanism by which PHA elicits its mitogenic action is un-
known.
P
out the brain and in peripheral tissues and is involved in a
broad spectrum of cellular functions (1). Lipid second mes-
sengers, produced in response to hormones, neurotransmit-
ters, or growth factors, regulate the activity of this enzyme.
An important outcome of cellular stimulation induced by
these substances is the translocation of PKC from cytosol to
membrane (2–6). PKC plays an important role in regulating
the release of neurotransmitters, such as acetylcholine (7),
norepinephrine (7), glutamate (8), and serotonin (3), as well
as in processes like synaptic potentiation that model various
aspects of learning and memory (9).
Both PKC activity and its translocation are reported to
change with advancing age in animal studies (3,5,10–11).
Consistent with these observations, we have observed an
age-dependent decrease in PKC activity and its transloca-
tion in postmortem human brains (12). Furthermore, we
have found significant reductions in platelet PKC activity
associated with both membranous and cytosolic cellular
fractions among elderly men, as well as in the redistribution
of platelet PKC activity that is elicited by stimulation of cell
surface receptors (13). We also observed, however, that
these age-related reductions were mitigated in older men
who had sustained moderately high levels of aerobic fitness
as they aged (13). Thus, PKC activity and its translocation
may be biological markers of aging that are sensitive to in-
terventions, such as physical exercise, that may serve to
slow the aging process.
MATERIALS AND METHODS
Subjects
Fifty-eight men, ranging in age from 20 to 36 and 61 to
78 years, participated in this study. With the exception of
one young nonexerciser and one older exerciser, this was
the same sample we evaluated in our previous study of
blood platelets (13). Subjects were identified on the basis of
self-report as sedentary nonexercisers or as regular aerobic
exercisers. For inclusion in the sample, young and older aer-
obic exercisers had to have trained systematically for a min-
imum of 2 or 10 years, respectively. These minimums were
set to insure that subjects were currently training regularly
and had done so for a reasonable period of time. The mini-
mum of 10 years for the older subjects was selected to in-
crease the likelihood that these subjects would have sus-
tained exercise for a long enough time to have derived
physiologic benefit. In practice, the participants in this
study, both young and older, had been exercising regularly
Age-related declines in lymphocyte-mediated immune
function, defined as the proliferative response to mitogenic
stimulation, have also been observed (14). This prolifera-
B545

B546
WANG ET AL.
for large portions of their lives: young and older subjects re-
ported exercising regularly for an average of 8.5 years
minutes. The supernatant contains extracted enzymes from
the membrane. Both cytosolic and membrane extracts were
applied to 1 ml of diethylaminoethyl cellulose (DE52, What-
man, Rockland, MA) equilibrated in buffer, and the columns
were washed, eluted with buffer containing 0.1 M NaCl,
and used immediately for assessing PKC activity.
(
range, 3–18 years) and 16.8 years (range, 10–40 years), re-
spectively. Four groups of subjects were thus formed: (i)
nine young nonexercisers, ranging in age from 23 to 34
years; (ii) 18 older nonexercisers, ranging in age from 62 to
7
8 years; (iii) 12 young exercisers, ranging in age from 20
The standard assay mixture (250 ␮l) containing 24 mM
Tris-HCl (pH 7.5), 20 mM NaCl, 0.1 mM EGTA, 0.4 mM
EDTA, 0.03% 2-mercaptoethanol, 60 ␮g/ml leupeptin, 0.04
mM phenylmethylsulfonyl fluoride, 0.25 mg/ml histone
to 36 years; and (iv) 19 older exercisers, ranging in age from
6
1 to 77 years.
Subjects were screened for medical exclusionary criteria
in a two-step process. A candidate first responded to a
newspaper advertisement or public announcement with a
telephone call during which a comprehensive medical his-
tory was taken. Individuals taking any medication that acts
on the central nervous system (CNS), reporting any current
or past medical disorder that could affect the CNS, having
histories of drug or alcohol abuse, or receiving nonsteroidal
antiinflammatory drugs that could interact with lympho-
cytes, were eliminated in this phase of the screening pro-
cess. Those candidates not excluded on the basis of these
criteria completed a medical examination that included a
history, physical, blood work, vision and hearing tests, and
an exercise stress test. The stress test was performed using a
stationary ergometer. It was stopped when the subjects
type III-s (Sigma, St. Louis, MO), 1.2 mM CaCl , 20 ␮g/ml
2
phosphatidyl-l-serine (PS), 80 nM PMA, 1 mM MgAc and
2
3
2
0.03 mM [ P]adenosine triphosphate (ATP) (DuPont, Bos-
ton, MA; 500,000 cpm) was preincubated at 30ЊC for 5 min-
utes, and the reaction was initiated by the addition of 50 ␮l
of eluted protein. After 1 minute, the reaction was termi-
nated by transferring 50 ␮l onto a 1 ϫ 2 cm phosphocellu-
lose (P81, Whatman, Rockland, MA) strip that was subse-
quently immersed in 75 mM phosphoric acid (10 ml per
strip). The strips were washed 3 times (2 min per wash) in
fresh phosphoric acid and air dried. They were then placed
in scintillation fluid and radioactivity was determined by
liquid scintillation spectrometry (LKB 1214 Rackbeta,
Stockholm, Sweden). PKC activity was defined as the phos-
phorylation that occurred in the presence of phosphatidyl-
l-serine (PS), and PMA and was expressed as pmol 32Pi
incorporated per unit protein of column eluate. Negligible
phosphorylation was measured when the column eluates
were assayed in the presence and absence of PS and PMA
without added substrate. Protein was determined by the
method of Lowry and colleagues. (16).
achieved 75% of predicted maximum heart rate. This yielded
.
an estimate of maximal oxygen consumption (VO max) that
2
provided an objective index of aerobic fitness to differenti-
ate exercisers from nonexercisers. Candidates who passed
the medical examination participated in the study. All par-
ticipants were free of clinical evidence of cardiovascular or
neurological illnesses or infectious diseases that could alter
lymphocyte functioning.
Statistical Analyses
PKC-Mediated Protein Phosphorylation and Enzyme
Redistribution in Lymphocytes
To maximize the quality of our data and thus the validity
of our analysis and interpretation, careful consideration was
given to model assumptions (and the consequences of viola-
tion of assumptions). Where violations were observed, steps
were taken to correct the data (or adjust the analysis). For
example, violations of normality assumptions were cor-
rected by data transformations. A general factorial analysis
of variance (ANOVA) (17), weighted for between-group
differences in sample size, was completed to determine the
Venous blood was collected into ethylenediaminetet-
raacetic acid (EDTA)-containing syringes (Luer-Monovette,
Sarstedt, Inc, Newton, NC). Lymphocytes were isolated us-
ing Sigma Histopaque-1077, according to the manufac-
turer’s instructions. The isolated lymphocytes were washed
in 10 ml of phosphate-buffered saline (PBS) prior to PKC
activity measurements, and then were incubated at 37ЊC in a
total of 0.5 ml for 10 minutes in 0.32 M sucrose buffered
with 5 mM N-2-hydroxyethylipiperazine-NЈ-2 ethanesulforic
acid (pH 7.5), with 80 nM PMA, or with 40 mg/ml PHA. The
concentrations of these agents were selected to approximate
their respective EC s for PKC activity and mitogenic action.
influence of the between-group factors for age and exercise
groups on the dependent measures: age (in years), VO max
.
2
(ml/min/kg), and cytosolic and membrane PKC activities in
lymphocytes. Significant main effects (p Ͻ .05) were fol-
lowed by post hoc multiple comparisons between means of
a given factor. For these post hoc mean comparisons,
Tukey’s honestly significant difference procedure (18) pro-
vided the appropriate balance between statistical power and
overall control of type I errors. Because this is an unbal-
anced design (number of cases in all the cells are not equal)
with no empty cells, we used a specific approach to calcu-
lating sums of squares (type III). This method calculates the
sum of squares of a specific effect in the design as the sum
of squares adjusted for any other effects that do not contain
it and orthogonal to any effects (if any) that do contain it.
Results of the Tukey test are only given when they added
information not provided by the tests of simple effects. The
degrees of freedom for all of the analyses were 1 and 54.
50
Reaction was stopped by the addition of 0.5 mM ethylene-
glycol-bis (␤-aminoethyl)-N,N,NЈ,NЈ-tetraacetic acid (EGTA)-
containing buffer and cooling on ice. Fresh lymphocytes
were harvested and homogenized in 1 ml, 0.32 M sucrose in
2
0 mM Tris-HCl (pH 7.5), with 2 mM EDTA, 0.5 mM
EGTA, 50 mg/ml leupeptin, 0.2 mM phenylmethylsulfonyl
fluoride, and 0.1% 2-mercaptoethanol and sonicated (Micro
Cell Disrupter, Kontes, Vineland, NJ) prior to centrifuga-
tion at 25,000 ϫ g for 15 minutes. The supernatant yielded
after centrifugation is considered the cytosolic fraction. The
pellet was resuspended in 200 ␮l of buffer containing 1%
Nonidet P-40 and solubilized on ice for 1 hour. The sample
was diluted to 1 ml and centrifuged at 25,000 ϫ g for 15

EXERCISE REDUCES AGE-RELATED DECREASE IN PKC
B547
Given this constancy in degrees of freedom across analyses,
when a statistical result is shown, it will include only the F
ratio and the exact p value (i.e., p ϭ .4358). In cases where
the p value is smaller than .0001, we will designate it as p Ͻ
Exercise Group interaction, [cyt] F ϭ 30.52, p Ͻ .0001;
[memb] F ϭ 27.07, p Ͻ .0001). Tests of the simple effects
revealed that young nonexercisers had higher cytosolic, but
not membrane-associated, enzyme activity levels than young
exercisers ([cyt] F ϭ 8.52, p ϭ .0088; [memb] F ϭ 3.18, p ϭ
.
0001.
.0904); however, older exercisers had higher levels of activ-
RESULTS
·
Age and VO max
2
.
Analyses on age and VO max were done to accomplish
2
two ends. We wanted to determine (i) if subjects within
each exercise group were matched on age and (ii) if subjects
who identified themselves as exercisers had higher levels of
measured aerobic fitness than subjects who described them-
selves as sedentary. Table 1 contains the ages and levels of
.
VO max for each of the four age-by-exercise groups. The
2
percentage of exercisers was slightly greater among the
young subjects (12 of 21) than among the older subjects (19
of 37). The possible confounding introduced by this small
imbalance was avoided by the use of type III sums of
squares in all two-way ANOVAs. Exercisers and nonexer-
cisers (Exercisers, 52.9 Ϯ 19.8, vs Nonexercisers, 57.0 Ϯ
2
0.2; Exercise Group, F ϭ 0.62, p ϭ .4358) differed in
.
.
VO max within each age group. As expected, VO max was
2
2
significantly higher in exercisers than in nonexercisers (44.1 Ϯ
0.0 vs 27.0 Ϯ 7.5; Exercise Group, F ϭ 53.36, p Ͻ .0001),
1
with the difference being larger among young versus older
subjects (significant Age Group ϫ Exercise Group interac-
tion, F ϭ 5.87, p ϭ .019; simple effects: young, F ϭ 64.86,
p Ͻ .0001; older, F ϭ 85.00, p Ͻ .0001).
Effects of Age and Exercise on Basal Lymphocyte
PKC Activity
A varied pattern of sensitivity to age and aerobic fitness
was evident for basal lymphocyte PKC activity. Age-sensi-
tive responses to aerobic fitness were apparent in both cyto-
solic and membrane-associated enzyme activities. Activity
levels in both cell fractions were significantly higher in
young than in older subjects (Age Group, [cyt] F ϭ 100.71,
p Ͻ .0001; [memb] F ϭ 62.96, p Ͻ .0001) and in exercisers
than in nonexercisers (Exercise Group, [cyt] F ϭ 5.23, p ϭ
.
0269; [memb] F ϭ 10.96, p ϭ .0027). However, as de-
picted in Figure 1, differences associated with aerobic fit-
ness in the activity levels of both measures varied among
the young and the older subjects (significant Age Group ϫ
·
Table 1. Mean Age and VO max, Age ϫ Exercise Groups
2
·
Group
n
Age (years)
VO
2
max
Young nonexercisers
9
29.0 Ϯ 3.8
34.4 Ϯ 7.3
(25.1–43.7)
23.3 Ϯ 4.3
(19.4–32.8)
55.4 Ϯ 4.4
(48.2–61.9)
37.2 Ϯ 4.9
(27.2–45.8)
(
23–34)
70.0 Ϯ 4.4
62–78)
29.6 Ϯ 4.8
20–36)
67.7 Ϯ 4.4
61–77)
Figure 1. Combined effects of age and aerobic fitness on protein ki-
Old nonexercisers
Young exercisers
Old exercisers
18
12
19
nase C (PKC) activity in lymphocyte cytosol and membrane fractions.
Lymphocytes from healthy young (Y) and older (O) nonexercisers
(NE) and exercisers (E) were collected and sonicated, and cytosolic
and membrane-associated PKC activities were determined. In this and
all subsequent figures, error bars are shown to indicate the standard
deviation, the absolute value for each measure for each group is given
in the bar accompanied by the standard deviation, and the absolute p
values are shown for the tests of the simple effects for the exerciser vs
nonexerciser comparison within an age group. The p values for the in-
teractions were Ͻ.0001 for both cytosol and membrane.
(
(
(
Notes: n ϭ number of subjects; values not in parentheses are the means Ϯ
standard deviation for each group; values in parentheses on the line below the
means are the range for the group.

B548
WANG ET AL.
ity on both measures than older nonexercisers ([cyt] F ϭ
7.90, p Ͻ .0001; [memb] F ϭ 45.91, p Ͻ .0001).
Table 2. PMA-Induced PKC Activation Ratios of
Membrane-to-Cytosol Activity, Age ϫ Exercise Groups
2
n
Young
n
Old
Effects of Age and Exercise on PKC Translocation
Induced by PMA
Nonexercisers
PMA
9
0.77 Ϯ 0.16
18
0.22 Ϯ 0.10
As noted previously, activation of PKC by the phorbol
ester, PMA, produces a redistribution of PKC activity from
cytosol to membrane. To evaluate the influences of age and
aerobic fitness on this redistribution, we stimulated PKC
with 80 nM of PMA. Although distribution of PKC in cyto-
solic and membranous fractions under basal conditions was
not altered by age or aerobic fitness (Age Group, F ϭ 0.61,
p ϭ .4219; Exercise Group, F ϭ 0.57, p ϭ .4314), both fac-
tors were associated with variations in the redistribution of
PKC following stimulation by PMA. These variations were
revealed in changes induced by PMA on the ratio of mem-
brane-to-cytosolic PKC activity, an index that reflects en-
zyme translocation. Young subjects had higher ratios than
older subjects (Age Group, F ϭ 56.89, p Ͻ .0001), and ex-
ercisers had higher ratios than nonexercisers (Exercise
Group, F ϭ 12.31, p ϭ .001). Although the direction of the
difference between the two age groups did not vary with
aerobic fitness, the Age Group ϫ Exercise Group interac-
tion was suggestive (F ϭ 2.33, p ϭ .1331). There is no ex-
tant literature, to our knowledge, that provides an a priori
basis to support any prediction regarding differences in the
magnitude of the combined influence of age and aerobic fit-
ness on PMA-induced changes in PKC activity. Inspection
of Figure 2 suggests, however, that differences in the mag-
nitude of the difference in PKC activity between exercisers
and nonexercisers may be larger among older than young
subjects. This impression was confirmed in tests of the sim-
ple effects. They showed that the ratios did not differ signif-
icantly among young subjects (F ϭ 0.56, p ϭ .4648),
whereas among older subjects, the ratio was significantly
higher among the aerobically fit (F ϭ 17.45, p ϭ .0002).
(
0.61–0.98)
(0.07–0.43)
Exercisers
PMA
12
0.86 Ϯ 0.31
0.43–1.44)
19
0.49 Ϯ 0.25
(0.17–1.09)
(
Notes: n ϭ number of subjects; values not in parentheses are the mean cyto-
sol-to-membrane ratios for each group Ϯ standard deviation; values in parenthe-
ses on the line below the means are the ranges for the group. PMA ϭ phorbol
1
2-myristate, 13-acetate; PKC ϭ protein kinase C.
Thus, PMA-induced differences in the translocation of PKC
between exercisers and nonexercisers were evident only
among older subjects. This set of ratios is shown in Table 2.
Effects of Age and Aerobic Fitness on Cytosolic and
Membranous PKC Activity Induced by PHA
The mitogen, PHA, increased lymphocyte PKC activity.
We evaluated the influence of age and aerobic fitness on this
enhancement by measuring the percent change in PKC activ-
ity induced by PHA in the four groups. These data are sum-
marized in Table 3 and illustrated graphically in Figure 3.
PHA induced a larger change in PKC activity among young
than older subjects in both cytosol and membrane fractions
(
Age Group: [cyt] F ϭ 45.52, p Ͻ .0001; [memb] F ϭ 26.07,
p Ͻ .0001). Likewise, it enhanced PKC activity to a greater
extent among exercisers than nonexercisers in both cytosol
and membrane (Exercise Group: [cyt] F ϭ 7.74, p ϭ .007;
[
memb] F ϭ 9.51, p ϭ .003). The influence of age on PHA-
induced PKC activity in cytosol varied, however, with aero-
bic fitness (significant Age Group ϫ Exercise Group inter-
action, F ϭ 5.53, p ϭ .0224; see Figure 3, top panel). Tests
of the simple effects showed that aerobic fitness did not in-
fluence the degree of PKC activation induced in cytosol by
PHA among young subjects (F ϭ 0.07, p ϭ .7939), but that
this activation was reduced among older nonexercisers
compared with exercisers (F ϭ 11.34, p ϭ .0019).
In contrast to the influence of aerobic fitness on age-related
changes in PHA-induced PKC activation in cytosol, aerobic
fitness did not appear to alter this activation differentially
between the two age groups (Age Group ϫ Exercise Group,
F ϭ 2.70, p ϭ .106). Again, however, as was the case for
PMA-induced changes in PKC activity, although there is no
a priori basis on which to predict a specific age-by-exercise
relationship, the interaction was suggestive and visual in-
spection of Figure 3 (bottom panel) reveals the possibility
of age-related differences. Thus, we completed tests of the
simple effects. They revealed that PHA-induced activation
of PKC in membrane was comparable among young nonex-
ercisers and exercisers (F ϭ 0.36, p ϭ .5531), but that it
was reduced in older nonexercisers compared with exercis-
ers (F ϭ 10.55, p ϭ .0026). Thus, differences between exer-
cisers and nonexercisers in PHA-induced activation of PKC
in both cytosol and membrane were evident only among
older subjects. Here it is of interest to note that young
nonexercisers and older exercisers did not differ in the
Figure 2. Combined effects of age and aerobic fitness on protein
kinase C (PKC) translocation induced by PMA in lymphocytes. Lym-
phocytes from healthy young (Y) and older (O) nonexercisers (NE)
and exercisers (E) were incubated for 10 min with 80 nM PMA, col-
lected and sonicated. Cytosolic and membrane-associated PKC activ-
ities were then determined. The p value for the interaction was .1331.

EXERCISE REDUCES AGE-RELATED DECREASE IN PKC
B549
Table 3. Percent Change in PHA-Induced Enhancement of PKC
Activity, Age ϫ Exercise Groups
n
Young
n
Old
Nonexercisers
PMAcyt
9
16.01 Ϯ 1.99
18
18
4.11 Ϯ 4.57
(0.00–14.50)
7.76 Ϯ 6.25
(0.00–25.30)
(13.10–18.80)
PHAmemb
9
22.20 Ϯ 5.42
(16.80–34.40)
Exercisers
PMAcyt
12
12
15.56 Ϯ 4.80
19
19
9.67 Ϯ 5.42
(Ϫ2.80–20.50)
16.25 Ϯ 9.95
(10.20–26.80)
PHAmemb
24.30 Ϯ 7.73
(14.50–44.30)
(Ϫ10.80–28.50)
Notes: n ϭ number of subjects; values not in parentheses are the mean
PHAcyt(osol) or PHAmemb(rane) for each group Ϯ standard deviation; values
in parentheses on the line below the means are the ranges for the group. PHA ϭ
phytohemagglutin; PKC ϭ protein kinase C.
PHA-induced activation of PKC in membrane (F ϭ 2.81,
p ϭ .1056).
DISCUSSION
We found that age induces declines among men in both
lymphocyte PKC activity and the activation of this enzyme
in response to cellular stimuli, but that the magnitude of
these declines is reduced by aerobic fitness. The levels of
aerobic fitness characteristic of the young and older exercis-
ers in this sample place them among the well conditioned
for their respective age groups, whereas the levels of aero-
bic fitness characteristic of the young and older nonexercis-
ers in this sample place them in the average range for their
respective age groups (see references in [19]). Hence the ex-
ercisers and nonexercisers in this sample represent individu-
als with very different levels of aerobic conditioning. It is of
interest to note that the age-dependent changes we found in
PKC activity (both cytosolic and membrane-associated) and
in the ability of the enzyme to translocate in response to
stimuli, parallel changes previously reported in animal stud-
ies (3,5,10–11). Thus, although maintenance of aerobic fit-
ness among older men may not preserve lymphocyte PKC
activity and enzyme redistribution at levels comparable
with those of the young, it may serve to slow the rate at
which enzyme activation declines with advancing age. This
observation accords well with a variety of other reports that
have demonstrated that older aerobically fit subjects tend to
fall between the young and the older aerobically decondi-
tioned on many measures that have been evaluated (19–23).
We observed an unexpected difference between young ex-
ercisers and nonexercisers on basal PKC activity in cytosol,
however; namely, young nonexercisers had higher levels
than young exercisers (whereas older exercisers had higher
levels than older nonexercisers). Given the overall pattern
of results in this and in our previous study of blood platelets
Figure 3. Combined effects of age and aerobic fitness on protein
kinase C (PKC) activation induced by PHA in lymphocytes. Lym-
phocytes from healthy young (Y) and older (O) nonexercisers (NE)
and exercisers (E) were incubated for 10 min with 40 mg/ml PHA,
collected and sonicated. Cytosolic and membrane-associated PKC ac-
tivities were then determined. The p values for the interactions for
cytosol and membrane were .0224 and .1060, respectively.
We have no explanation for this difference, but are inclined
to accept the latter explanation.
The activation of PKC seen in association with its trans-
location to membrane in response to cellular stimuli is de-
pendent on membrane constituents, such as phospholipids
2ϩ
(
13), we expected no differences in baseline activity be-
and phospholipase C, and on the availability of Ca . Thus,
alteration in the metabolism of membrane phospholipids,
changes in Ca2 homeostasis, and changes in membrane
fluidity during senescence may have a significant impact on
the activation of PKC in lymphocytes (24–27). Alterna-
tween the two young groups. This variation may reflect an
important physiological difference between the two groups
or it may simply reflect a statistical artifact emanating from
the relatively small sample sizes of the two young groups.
ϩ

B550
WANG ET AL.
tively, posttranslational modification of the enzyme, such as
phosphorylation or changes in the relative expression of
PKC isozymes in peripheral blood T and B lymphocytes
Acknowledgments
This work was supported by the US Public Health Service (Grants AG-
0
7700, MH-40380, AG-00131, AG-04581) and an American Federation for
Aging Research grant-in-aid (1989). We thank Dr. Kevin K. McCully and
two anonymous reviewers for their thoughtful comments on a previous ver-
sion of this article. We also thank Dr. Philip Weiser, Dr. Lee Greenspon,
and Dr. Joel Posner for performing the exercise stress tests. We owe a spe-
cial thanks to Ms. Carolann Imbesi for her help in preparing the manuscript.
(
28), may be responsible for the age-associated changes in
the activation and translocation of PKC. In addition, recent
data also indicate that age-dependent changes in the recep-
tors for activated C kinase, or RACKs, may be responsible
for some portion of the age-related decrease in PKC activa-
tion (29). It is possible that the differential activation of
PKC isozymes in lymphocytes may be associated with age-
related changes in immunological responses (14,30–32).
This possibility is supported by data that show that an in-
crease in ␣PKC level is coupled with a decrease in the pro-
liferative response of splenic T cells (33).
Address correspondence to Eitan Friedman, PhD, Department of Pharma-
cology and Physiology, MCP Hahnemann School of Medicine, Mailbox 488,
245 N. 15th Street, Philadelphia, PA 19102. E-mail: eitan.friedman@drexel.edu
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turnover and Ca -dependent PKC activation, has been
found to be associated with an age-related decline in T cell
function (32). Diminished diacylglycerol level, another
product of phosphoinositide metabolism, may result in a de-
crease in PKC activity and translocation, as has been previ-
ously suggested in other cell types (11). More importantly,
our data indicate that the age-related reduction in PKC ac-
tivity and its intracellular redistribution in response to stim-
ulation can be reduced in magnitude by aerobic fitness. Al-
though the precise mechanisms by which aerobic fitness
modifies physiological functions are not currently known,
the present results, together with previous observations in
other systems (35–38), may reflect a contribution aerobic
fitness makes to maintaining functions that would otherwise
decline at a faster rate with advancing age. The effects of
aerobic exercise in reducing the age-dependent decrease in
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pholipid content and/or in Ca homeostasis that are known
to be affected by exercise (39–40).
Thus, the data we have presented suggest once again that
sustained aerobic fitness may have beneficial effects on
changes in basic biological processes that are induced by
advancing age. We should underscore, however, that our
sample was restricted to men. Hence, the differences we
have reported here and in our earlier study (13) may not be
generalizable to women. In addition, because our compari-
sons were only of young and older adults, we cannot draw
any inferences about the timing of changes in PKC activity
over the lifespan, about critical periods, if any, when these
changes may express themselves, or about when in the
lifespan the influence of aerobic fitness on PKC activity
may be optimal. For example, because the older exercisers
in our study had typically exercised most of their adult lives,
we cannot conclude that initiation of aerobic activities later
in life will produce changes in PKC activity like those we
have found. We are left, then, with a variety of interesting
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Received October 21, 1998
Accepted March 30, 2000
Decision Editor: Jay Roberts, PhD